Oils enriched with diacylglycerols and phytosterol esters and unit dosage forms thereof for use in therapy

ABSTRACT

Described herein are various lipid mixtures, more particularly special mixtures of fatty acids esters, which may be phytosterol esters and/or glyceride esters. Said mixtures are evaluated in the context of their ability to, upon consumption, affect the levels of circulating LDL and HDL particles, and their therapeutic effect on conditions associated with lipid metabolism, such as atherosclerosis, diabetes, metabolic disorders, etc. Compositions, dietary nutrients, food supplements and nutraceuticals comprising the herein described mixtures are also described.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation in Part of U.S. patent applicationSer. No. 11/199,584 filed Aug. 8, 2005, which is a Continuation in Partof International Patent Application No. PCT/IL2004/000131 filed Feb. 10,2004.

FIELD OF THE INVENTION

The present invention relates to various lipid mixtures, moreparticularly special mixtures of fatty acids esters, which may bephytosterol esters and/or glyceride esters, as well as compositions,dietary nutrients, food supplements and nutraceuticals comprisingthereof, intended for the treatment and prevention of diseases andconditions associated with lipid metabolism.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fullyincorporated herein by reference, including all references citedtherein.

Coronary Artery Disease (like atherosclerosis) is the major cause ofmorbidity and mortality in the Western world and its pathogenesisinvolves complicated interactions between cells of the arterial wall,blood cells, and plasma lipoproteins [Ross R. (1993) Nature 362:801-809; Glass C. K. and Witztum J. L. (2001) Cell 104:503-516]. Today,it is common knowledge that lowering cholesterol levels reduces the riskof heart attacks, strokes and other forms of atherosclerotic vasculardisease. In addition, many recent studies have shown that oxidativestress is a mechanism with a central role in the pathogenesis ofatherosclerosis, cancer, and other chronic diseases, e.g. diabetes. Inthis scenario, a key role is played by macrophages in thesub-endothelial space, which are activated by oxidized low-densitylipoproteins (ox-LDL). Recently, endothelial dysfunction due tooxidative stress was identified as a priming factor in the course of thedevelopment of atherosclerotic plaques.

LDL-cholesterol (LDL-C) levels are currently recommended as the primarytarget for lipid lowering therapy for prevention of CVD. The role ofLDL-C in the development of atherosclerosis, the relation between bloodLDL-C levels and risk of CVD, and the beneficial effects of LDL-Clowering therapy are well established. Similarly, it is well known thatlow levels of HDL cholesterol (HDL-C) are associated with an increasedrisk for CVD independent of LDL-C levels, and that raising HDL-C hasbeen shown to significantly lower CVD risk. Total cholesterol/HDL-Cratio is most predictive of CHD as was demonstrated in severalretrospective clinical trials. However, other factors may significantlyaffect the risk for CHD, and there is a growing body of evidencestrongly suggesting a role of plasma triglycerides concentrations as anindependent risk factor of CHD [Assmann G., et al. (1996) Am. J.Cardiol. 77(14):1179-1184]. Omega-3 fatty acids influence CVD risk in amultifactorial manner. Numerous findings, including evidence fromrandomized controlled trials, demonstrated the beneficial effects ofOmega-3 long-chain polyunsaturated fatty acids (LC-PUFA) on CVD risk inpatients with preexisting CVD as well as in healthy individuals.Large-scale epidemiological studies suggest that individuals at risk forCHD benefit from the consumption of plant- and marine derived Omega-3fatty acids. Evidence from prospective secondary prevention studies andrandomized controlled trials indicate that Omega-3 fatty acidssupplements can reduce cardiac events (e.g., death, non-fatal myocardialinfarct, non-fatal stroke) and decrease progression of atherosclerosisin coronary patients [Kris-Etherton P M., et al. (2002) Circulation106(21):2747-2757] Omega-3 fatty acids reduce very-low densitylipoprotein (VLDL) secretion, lower triglycerides transport and enhanceVLDL clearance, and reduce circulating triglycerides. Omega-3 fattyacids have markedly anti-inflammatory, anti-thrombotic andimmuno-modulatory properties that may be beneficial in CVD.

EPA and DHA may alter HDL cholesterol subclasses. Increases in the HDL₂subfraction have been reported with supplementation of 4 g DHA/d inhyperlipidemic men and type 2 diabetic patients [Mori, T. A. et al.(2000) Am. J. Clin. Nutr. 2000; 71: 1085-94]. The effect of EPA on HDL₂subclasses is less clear; a lowering effect on HDL₃ concentrations, withno effect on HDL₂ have been observed. When supplemented simultaneously,1.48 mg DHA and 1.88 mg EPA/d were shown to increase HDL₂ concentrationsin subjects with familial combined hyperlipidemia, a disordercharacterised by low HDL₂ concentrations [Calabresi, L. et al. (2004)Metabolism 53:153-8]. HDL cholesterol concentrations are usually notsignificantly affected by plant sterols, but a slight increase has beenreported in a few studies [Gylling, H. and Miettinen, T. A. (1999)Metabolism 48: 575-80].

Subjects identified as having low density lipoprotein (LDL) cholesterolconcentrations above 130 mg/dl are routinely counselled to modify theirdiet with respect to saturated fat and cholesterol intake. At presentthe dietary guidelines are relatively broad; total fat 20-25% of energy(<10% of energy saturated fatty acids [SFA], 5-15% of energymonounsaturated fatty acids [MUFA], up to 10% of energy polyunsaturatedfatty acids [PUFA], <300 mg cholesterol per day). In addition to theaforementioned dietary recommendations, the use of plant sterols andstanols to optimize blood lipid levels has gained increased importancethrough the year 2001, with the new recommendation released from theNational Cholesterol Education Program advising the public to consume 2g per day of plant sterols or stanols in addition to the TherapeuticLifestyle Change Diet to lower elevated LDL cholesterol levels. Plantsterols and stanols are now widely available in many countries acrossthe world as functional foods possessing government-approved healthclaims.

Paraoxonase (PON1) is an esterase, transported in the plasma as acomponent of HDL, associated to ApoAI and ApoJ. It has been shown invitro that purified PON1 and HDL-associated PON1 inhibit the oxidativemodification of LDL. Thus, the presence of PON1 in HDL may account for aproportion of the anti-oxidant properties of these lipoproteins[Tsuzura, S., et al. (2004) Metabolism 53:297-302]. Interestingly,several investigators have shown that serum paraoxonase activity islower in diabetic patients and is lower yet in those with diabeticcomplications, independent of PON1 gene polymorphisms. Theseobservations are consistent with in vivo increased oxidative stresslevels in diabetic patients.

The LDL oxidation hypothesis of atherosclerosis raised an extensiveinvestigation into the role of anti-oxidants against LDL oxidation as apossible preventive treatment of atherosclerosis. Although increasedresistance of LDL to oxidation was observed after treatment with varioussynthetic pharmaceutical agents, an effort has been made to identifynatural food products, which offer anti-oxidant defense against LDLoxidation.

Olive oil has been shown to inhibit LDL oxidation and this effect couldbe related to its high oleic acid content, as well as to some phenolics(hydroxytoluene, oleoropein) and phytosterols such as sitosterol [AviramM. and Kasem E. (1993) Ann. Nutr. Metabol. 37:75-84; Visioli F. et al.(1995) Atherosclerosis 117:25-32].

In addition to LDL oxidation, a known risk factor for coronary heartdisease (CHD)—the result of atherosclerosis in the coronaryarteries—includes high serum LDL cholesterol concentration. There is apositive linear relationship between serum total cholesterol and LDLcholesterol concentrations, and risk of, or mortality from CHD[Jousilahtu et al. (1998) Circulation 97:1084-1094].

High concentrations of serum triacylglycerols may also contribute to CHD[Austin, M. A. (1989) Am. J. Epidemiol. 129:249-259], but the evidenceis less clear. Diacylglycerols (DAG) have been shown to lower thepostprandial elevation of serum triacylglycerols levels compared withtriacylglycerols in healthy humans [Taguchi, H et al. (2000) J. Am.Coll. Nutr. 19:789-796].

Phytosterols and CHD

The term “phytosterols” covers plant sterols and plant stanols,including beta-sitosterol, campesterol and stigmasterol. Plant sterolsare naturally occurring substances present in the diet as minorcomponents of vegetable oils. Plant sterols have a role in plantssimilar to that of cholesterol in mammals, e.g. forming cell membranestructures. In human nutrition, both plant sterols and plant stanols areeffective in lowering total plasma cholesterol levels andLDL-cholesterol.

The consumption of plant sterols and plant stanols lowers bloodcholesterol levels by inhibiting the absorption of dietary andendogenously-produced cholesterol from the small intestine. The plantsterols/stanols are very poorly absorbable compounds. This inhibition isrelated to the similarity in physico-chemical properties of plantsterols and stanols to cholesterol.

In addition, both plant sterols and plant stanols have been subjected torigorous toxicological evaluation. Studies on the absorption,distribution, metabolism and excretion have shown that plant sterols arepoorly absorbed from the intestine (1-10%).

The specific plant sterols that are currently incorporated into foodsfor their hypocholesterolemic effects are extracts of soybean or tall(pine tree) oils. In most cases these plant sterols are esterified tounsaturated fatty acids (creating sterol esters) to increase miscibilitywithin the foods they are normally matrixed into. Some plant sterolscurrently in use are hydrogenated prior to esterification, resulting insaturated stanol derivatives, or plant stanols, such as beta-sitostanoland campestanol. Additional sources of plant sterols that are nowbecoming available are derived directly from corn fiber and not furthermodified, microcrystallinized in a way that obviates the need foresterification, or esterified to specific fatty acids that may haveindependent biological activity such as omega-3 fatty acids.

In the early 1950's plant derived sterols were first observed todecrease serum cholesterol levels and were marketed by Eli Lilly asCytellin™.

Plant sterols consumption at 2-3 g/day has been demonstrated to lowercirculating LDL cholesterol levels by 10-15% in humans withhyperlipidemia, offering a useful dietary strategy to risk managementfor heart disease. The mechanism of action of plant sterols in loweringLDL levels remains to be fully explained, however, it has beendemonstrated that plant sterols act by excluding dietary and biliarycholesterol from micelles in the intestine during the process ofabsorption. Plant sterols, due to their chemical structure, preventcholesterol from entering the outer micellar zone, thereby restrictingthe passage of cholesterol from the intestinal lumen across into themucosal cell of the intestinal wall. Recently, it has been demonstratedthat certain types of compounds, such as ascorbic acid, attached to thehydroxyl group of plant sterols such as sitosterol and campesterol asesters, increase the efficacy of cholesterol lowering in animalexperiments, likely through more aggressive exclusion of cholesterolfrom the micelle.

In the United States, a panel of independent experts has concluded thatvegetable oil sterol esters, meeting appropriate food-gradespecifications and produced by current good manufacturing practice (21C.F.R. §182.1(b)), are safe for use as an ingredient in vegetable oilspreads, in amounts which do not exceed 20% of plant sterol esters. Itwas the Panel's opinion, together with qualified experts in the field,that vegetable oil sterol esters are safe for use, i.e. vegetable oilsterol esters were granted the GRAS status (Generally Recognized AsSafe). Based on the GRAS recognition, the US Food and DrugAdministration (FDA) has cleared to use a spread containing up to 20% ofplant sterol esters and another one containing plant stanol ester.Similar approvals were given in different European countries as well asin Asia and Australia.

A recent review teaches that in recent years, with the growing interestin functional foods, the use of phytosterols for reducing serumcholesterol levels has gained considerable momentum [Stark, A. H. et al.(2002) Nutrition Reviews 60(6):170-176]. This should be attributed,inter alia, to the esterification of phytostanol with fatty acids(stanyl esters), providing commercial scale production ofphytosterol-containing foods, such as margarines. Like stanyl esters,phytosteryl esters (steryl esters) have been shown in clinical studiesto consistently lower serum LDL-cholesterol (LDL-C) levels (reducing byup to about 10% or more), with no change seen in HDL-cholesterol (HDL-C)values. The review suggests that properly formulated free phytosterolsand stanols may be as effective as stanyl and steryl esters in loweringLDL-C levels in humans.

WO 01/32035 teaches olive oil-based products, based on especially highergrades of olive oils (such as virgin olive oils), comprising plantstanol esters and/or plant sterol esters.

U.S. Pat. No. 5,843,499 discloses oil extractable from corn fiber thatcontains ferulate esters (phytosterol esters which are esterified toferulic acid), in particular sitostanyl ester, which has been shown tohave cholesterol-lowering activity. It is mentioned that corn fiber oil(containing about 73% fat (triacylglycerol), 8% sterol (fatty acyl)esters, 4% free sterols, 6% diacylglycerols and 6% ferulate (sterolesters)) is used as an additive to supplementary food for reducingcholesterol level.

U.S. Pat. No. 6,326,050 discloses a composition consisting of oil orfat, a diacylglycerol, a free phytosterol and tocopherol, dissolved ordispersed in the oil or fat. This composition plays a role in loweringblood cholesterol of hypercholesterolemic individuals.

However, none of the above mentioned publications describes reduction ofboth cholesterol and triglycerides serum levels.

Olive oil, in contrast to other mentioned vegetable oils (such as cornfiber oil, table cooking oil, soybean oil, rapeseed oil, rice bran oil,and palm oil) is composed, inter alia, of 55 to 85% monounsaturatedfatty acids (MUFA), in particular oleic acid, which contribute to thehigh nutritional value of this oil. There are some distinct advantagesof using olive oil over other vegetable oils. Diets rich in olive oilhave been shown to be more effective in lowering total cholesterol andLDL-cholesterol than conventional dietary treatments not containing highlevels of MUFA [Brown M. S and Goldstein J. L. (1983) Ann. Rev. Biochem.52:223-261].

Furthermore, olive oil is an integral ingredient of the Mediterraneandiet and accumulating data suggests that it may have health benefitsthat include reduction of risk factors of coronary artery disease,prevention of several types of cancer, and modification of immune andinflammatory response [Brown and Goldstein (1983) id ibid.].

WO01/15552 describes a nutritional supplement comprising purified estersof omega-3 fatty acids with phytosterols, for lowering triglyceride andcholesterol blood levels. This publication does not describe mixtures ofsuch esters with a fat base, such as fish oil. Moreover, thispublication does not describe any effect of the disclosed esters onblood lipids sub-fractions.

U.S. Pat. No. 6,589,588 discloses a sterol or stanol composition,wherein the fatty acid moiety comprises a blend of less that 7% ofsaturated fatty acids and more than 50% of polyunsaturated fatty acids,for lowering absorption of cholesterol for the digestive tract. Alsothis publication does not describe mixtures of the esters with fish oil,or any other fat, oil or lipid, and does not present any results as tothe cholesterol reducing activity of the esters, neither any otheractivity, such as lowering triglycerides, controlling HDL subfractions,or the like.

Co-owned WO03/064444 describes a composition of matter comprisingdiacylglycerol(s), mainly 1,3-diacylglycerols (DAG) and phytosteroland/or phytostanol ester(s) (PSE), dispersed in oil and/or fat.

In the parent application, U.S. Ser. No. 11/199,584, the inventorsreport that said combination of diacylglycerol(s), mainly 1,3-DAGs, andPSEs, preferably dissolved or dispersed in oil and/or fat, has asynergistic effect and decreases both LDL-cholesterol and triglycerideslevels in the blood. This combination further exhibited increased serumand macrophage anti-oxidative properties, and in particular LDLanti-oxidative properties, resulting in reduction of the risk for CHDand arteriovascular diseases.

In the parent application, an effect in reducing both cholesterol andtriglycerides serum levels, together with increased anti-oxidativeproperties, was observed even when a combination containing only 11 wt %DAG and 20 wt % phytosterol esters (in oil) was employed.

It is an object of the present invention to provide mixtures of omega-3fatty acids, preferably DHA and EPA, esterified to other lipids withimproved activity in, e.g. reducing levels of as apolipoprotein B,decreasing levels of HDL subfraction HDL₃, whilst elevating the level ofHDL₂.

These and other objects of the invention will become apparent as thedescription proceeds.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides an edible mixture ofesters of long chain polyunsaturated fatty acids (LC-PUFA), wherein saidmixture comprises esters of LC-PUFA with phytosterols and/orphytostanols and esters of LC-PUFA with glycerol, optionally alsocomprising free phytosterols and/or phytostanols, wherein the weightratio of said LC-PUFA glycerol esters to their esters with phytosterolor phytostanol is from about 19:1 to about 1.5:1. In one preferredembodiment, said esters of LC-PUFA with glycerol are mono-, di- and/ortriglycerides.

In a second aspect the present invention provides an edible compositionof matter comprising a mixture of esters of LC-PUFA with glycerol,enriched with a mixture of esters of LC-PUFA with phytosterols and/orphytostanols, wherein said mixture of esters LC-PUFA with glycerol isderived from: an animal source, particularly fish oil, a plant, algae ormicroorganisms; and wherein the weight ratio of said LC-PUFA glycerolesters to their esters with phytosterol or phytostanol is from about1.5:1 to about 1:9.

The mixture described herein, or the edible composition of mattercomprising thereof, is intended for various uses, including:

(a) reducing the risk of developing metabolic disorder and/or insulinresistance syndrome;

(b) reducing circulating atherogenic small-dense LDL particles, whilemaintaining a high proportion of HDL/LDL ratio;

(c) shifting the HDL profile in the direction of larger and less denseparticles, particularly reducing the HDL₃ sub-fraction while increasingthe level of HDL₂ sub-fraction;

(d) reducing and/or preventing the progression of the metabolic disorderand/or insulin resistance syndrome and/or atherosclerotic deleteriousprocesses;

(e) attenuating or counteracting any adverse effects of use of omega-3fatty acids in the form of oils, wherein said adverse effect may beelevating LDL cholesterol or increasing the total/HDL cholesterolration, related to a decrease in HDL cholesterol levels.

In a further aspect the present invention provides an orallyadministrable pharmaceutical or nutraceutical unit dosage formcomprising an edible mixture of esters LC-PUFA with glycerol, enrichedwith a mixture of esters of LC-PUFA with phytosterols and/orphytostanols, wherein said unit dosage form contains about 1.4-1.6 g ofsaid phytosterol/phytostanol LC-PUFA esters, said dosage form beingcapable of providing at least 50% of the recommended daily intake (RDI)of phytosterols and the recommended daily intake of LC-PUFA. Preferably,said RDI of phytosterols is from about 0.4 g to about 0.8 g and said RDIof LC-PUFA, specifically, DHA and EPA, is from about 0.3 g to about 0.65g.

In one preferred embodiment said pharmaceutical dosage form is in theform of a capsule, preferably a soft gel capsule.

Similarly, said pharmaceutical dosage form is intended for the same usesas described above for the mixture of the invention, i.e., in reducingthe risk of developing metabolic disorder and/or insulin resistancesyndrome; in reducing circulating atherogenic small-dense LDL particles,while maintaining a high proportion of HDL/LDL ratio; in shifting HDLprofile in the direction of larger and less dense particles,particularly reducing the HDL₃ subfraction while increasing the level ofHDL₂ subfraction; in reducing and/or preventing the progression of themetabolic disorder and/or insulin resistance syndrome and/oratherosclerotic deleterious processes; in attenuating or counteractingany adverse effects of use of omega-3 fatty acids in the form of oils,wherein said adverse effect may be elevating LDL cholesterol orincreasing the total/HDL cholesterol ration, related to a decrease inHDL cholesterol levels.

In a further most preferred embodiment, the pharmaceutical dosage formdescribed herein above is for twice daily administration.

In a yet further aspect, the present invention provides a method oftreatment of metabolic syndrome and related conditions, said methodcomprising administering a therapeutically effective dose of an ediblemixture of esters of LC-PUFA or compositions comprising thereof to asubject in need, wherein said mixture comprises esters of LC-PUFA withphytosterols and/or phytostanols and esters of LC-PUFA with glycerol,wherein the weight ratio of said LC-PUFA glycerol esters to their esterswith phytosterol or phytostanol is from about 19:1 to about 1:9.

In an even further aspect, the present invention provides a method ofreducing circulating atherogenic small-dense LDL particles, whilemaintaining a high proportion of HDL/LDL ratio, said method comprisingadministering a therapeutically effective dose of an edible mixture ofesters of LC-PUFA or compositions comprising thereof to a subject inneed, wherein said mixture comprises esters of LC-PUFA with phytosterolsand/or phytostanols and esters of LC-PUFA with glycerol, wherein theweight ratio of said LC-PUFA glycerol esters to their esters withphytosterol or phytostanol is from about 19:1 to about 1:9.

In a final aspect the present invention provides a method of shiftingthe HDL profile in the direction of larger and less dense particles,particularly reducing the HDL₃ subfraction while increasing the level ofHDL₂ subfraction, said method comprising administering a therapeuticallyeffective dose of an edible mixture of esters of LC-PUFA or compositionscomprising thereof to a subject in need, wherein said mixture comprisesesters of LC-PUFA with phytosterols and/or phytostanols and esters ofLC-PUFA with glycerol, wherein the weight ratio of said LC-PUFA glycerolesters to their esters with phytosterol or phytostanol is from about19:1 to about 1:9.

Common to all methods described above, said effective dose is preferablycomprised in a soft gelatine capsule, while said method comprisesadministering said capsule to said patient twice daily.

In addition, it is to be understood that the weight ratio of saidLC-PUFA glycerol esters to their esters with phytosterol or phytostanolcomprised in the mixture used in the methods described above may also bein the range between 9:1 to 1:9, or from 9:1 to 1:3, or more preferablyfrom 3:1 to 1.5:1.

The invention will be described in more detail on hand of theaccompanying Figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B: The effect of olive oil, olive oil+phytosterols, and PSE+DAGin olive oil on macrophage cellular peroxides content

FIG. 1A: Macrophage peroxide levels determined by the cells meanfluorescence (emitted by DCF) intensity.

FIG. 1B: Macrophage peroxide levels determined by the percentage offluorescent positive cells.

Abbreviations: PS-E, Plant sterols esters; DAG, diacylglycerol, cont.,control; Ol. O., olive oil.

FIG. 2: The effect of olive oil, olive oil+phytosterols, and PSE+DAG inolive oil on macrophage superoxides anions release

Macrophage superoxide ions release was determined by the superoxidedismutase-inhibitable reduction of cytochrome C.

Abbreviations: PS-E, Plant sterols esters; DAG, diacylglycerol, cont.,control;

Ol. O., olive oil.

FIG. 3: Effect of PSE+DAG in canola oil consumption on serumtriglycerides profile

FIG. 4: Effect of PSE+DAG in canola oil consumption on serum totalcholesterol profile

FIG. 5: Effect of PSE+DAG in canola oil and PSE+DAG in fish oilconsumption on serum oxidative stress

FIG. 6: Effect of PSE+DAG in canola oil and PSE+DAG in fish oilconsumption on serum PON1 activity

Abbreviations: Ser. PON1 act., serum PON1 activity; Plac., placebo;Can., canola.

FIG. 7: Effect of PSE+DAG in canola oil and PSE+DAG in fish oilconsumption on ox-LDL uptake by peritoneal macrophages

Abbreviations: deg., degradation; ass., association.

FIG. 8: Effect of PSE+DAG in canola oil and PSE+DAG in fish oilconsumption on macrophage oxidative status

FIG. 9: Effect of PSE+DAG in canola oil and PSE+DAG in fish oilconsumption on PMA-induced superoxide anions release in macrophages

FIG. 10: Change in total cholesterol concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol olive oil diet or PS-E+DAG in olive oil followed by four weeksof washout and counter supplementation. Total cholesterol levels weretested at the beginning (open squares) and at the termination (closedsquares) of each phase as described in methods. Values representmean±SEM of the total cholesterol concentrations in 21 patients.Statistical significance between baseline and endpoint values as foundby Student's t-test is * P<0.05 or ** P<0.001.

FIG. 11: Change in LDL cholesterol concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol olive oil diet or PS-E+DAG in olive oil followed by four weeksof washout and counter supplementation. LDL cholesterol levels weretested at the beginning (open squares) and at the termination (closedsquares) of each phase as described in methods. Values representmean±SEM of the LDL cholesterol concentrations in 21 patients.Statistical significance between baseline and endpoint values as foundby Student's t-test is * P<0.10 or **P<0.001.

FIG. 12: Change in Apolipoprotein B concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol olive oil diet or PS-E+DAG in olive oil followed by four weeksof washout and counter supplementation. ApoB levels were tested at thebeginning (open squares) and at the termination (closed squares) of eachphase as described in methods. Values represent mean±SEM of the apoBconcentrations in 21 patients. Statistical significance between baselineand endpoint values as found by Student's t-test is * P<0.05 or**P<0.001.

FIG. 13: Change in ApoB to ApoA ratio

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol olive oil diet or PS-E+DAG in olive oil followed by four weeksof washout and counter supplementation. ApoB/ApoA ratios were tested atthe beginning (open squares) and at the termination (closed squares) ofeach phase as described in methods. Values represent mean±SEM of theApoB/ApoA ratios in 21 patients. Statistical significance betweenbaseline and endpoint values as found by Student's t-test is * P<0.05.

FIG. 14: Change in lipoprotein(a) concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol olive oil diet or PS-E+DAG in olive oil followed by four weeksof washout and counter supplementation. Lp(a) levels were tested at thebeginning (open squares) and at the termination (closed squares) of eachphase as described in methods. Values represent mean±SEM of the Lp(a)concentrations in 21 patients. Statistical significance between baselineand endpoint values as found by Student's t-test is * P<0.01.

FIG. 15: Change in serum lipid peroxides concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol olive oil diet or PS-E+DAG in olive oil followed by four weeksof washout and counter supplementation. TBARS levels were tested at thebeginning (open squares) and at the termination (closed squares) of eachphase as described in methods. Values represent mean±SEM of the TBARSconcentrations in 21 patients. Statistical significance between baselineand endpoint values as found by Student's t-test is * P<0.10 or**P<0.01.

FIG. 16: Change in total cholesterol concentrations.

Preparation A vs. Fish oil vs. control.

FIG. 17: Change in LDL-cholesterol concentrations.

Preparation A vs. Fish oil.

FIG. 18: Change in apoB concentrations.

Preparation A vs. Fish oil vs. control.

FIG. 19: Change in apoA/apoB ratio.

Preparation A vs. Fish oil vs. control.

FIG. 20: Change in total to HDL cholesterol ratio.

Preparation A vs. Fish oil vs. control.

FIG. 21: Changes in HDL cholesterol sub-fractions.

Preparation A vs. Fish oil vs. control.

FIG. 22: Change in fasting triglycerides concentrations.

Preparation A vs. Fish oil vs. control.

FIG. 23: Endpoint postprandial triglycerides concentrations.

Preparation A vs. Fish oil vs. control.

FIG. 24: Change in CRP concentrations.

Preparation A vs. Fish oil vs. control.

FIG. 25: Endpoint relative risk according to Total/HDL cholesterol andCRP concentrations.

Preparation A vs. Fish oil vs. control.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations and definitions are used along thespecification:

-   AI: Acceptable intake-   ATP III: Adult Treatment Panel III (also appears as ATP 3)-   Canola composition: PSE+DAG in a canola oil base.-   CHD: Coronary heart disease-   CRP: C-reactive protein-   CVD: Cardiovascular disease-   DAG: Diacylglycerol(s), mainly 1,3-diacylglycerols-   DCF Dichlorofluorescin-   DHA: Docosahexaenoic acid-   EPA: Eicosapentaenoic acid-   Fish oil composition: PSE+DAG in a fish oil base.-   HBSS: Hanks' Balanced Salts Solution-   HDL: High density lipoproteins-   HDL-C: HDL cholesterol-   IHD: Ischemic heart disease-   LC-PUFA: Long chain PUFA-   LDL: Low density lipoproteins-   LDL-C: LDL cholesterol-   MPM: Mouse peritoneal macrophages-   MUFA: Monounsaturated fatty acid-   Olive oil composition: PSE+DAG combination in an olive oil base.-   Ox-LDL: Oxidized LDL-   PBS: Phosphate Buffered Saline-   PE: Phytosterol ester-   PMA: Phorbol myristate acetate-   PSE: Phytosterol or phytostanol ester(s)-   PSE+DAG combination: A combination of diacylglycerol(s) (DAG),    mainly 1,3-diacylglycerol(s) and phytosterol and/or phytosterol    esters (PSE)-   PSE+DAG composition: A composition of diacylglycerol(s) (DAG),    mainly 1,3-diacylglycerol(s) and phytosterol and/or phytosterol    esters (PSE) in an oil and/or fat base.-   PUFA: Polyunsaturated fatty acids-   RDA: Recommended daily allowance.-   RDI: Recommended daily intake-   TG: Triglyceride/s-   VLDL: Very low density lipoproteins

The present invention relates to various lipid-soluble agents, dietarynutrients, food supplements and nutraceuticals, intended for thetreatment and prevention of diseases and conditions associated withlipid metabolism.

In the present invention, the active agents are primarily mixtures ofesters of LC-PUFA, the esters being phytosterol and/or phytostanolesters, together with esters of the LC-PUFA with glycerol, which may beLC-PUFA mono-, di- and/or triglycerides.

The present inventors used the animal model system apoE⁰ mice, in whichsevere hypercholesterolemia and atherosclerotic plaques are generated atan early age to evaluate the anti-atherosclerotic properties of noveledible compositions, herein referred to as PSE+DAG-(olive),PSE+DAG-(canola) and PSE+DAG-(fish), in comparison with placebo and/orwith canola oil.

As mentioned above, the inventors found that a combination of DAGs,mainly 1,3-DAGs and PSE in oil and/or fat, provides an enhanced effect,by decreasing both LDL-cholesterol and triglycerides levels in theblood. This combination, and compositions comprising the same, furtherexhibits increased serum, serum LDL and macrophage anti-oxidativeproperties, as well as inhibiting the formation of foam cells and/orpreventing the deleterious effects generated by lipid-induced oxidativestress, which result in reduction of the risk for CHD andarteriovascular-related diseases, like, e.g., diabetes. These resultswere confirmed in a human study, as presented below.

While some mixtures of the kind have been described before, the presentinventors have now found that such known mixtures have specific finelytuned activity in lowering the blood levels of various particularlipids, and can thus be used for the treatment or prevention of medicaland physiological conditions associated with pathologically orabnormally increased levels of such lipids, or elevate levels of otherlipids, and thus be used in the treatment or prevention associated withpathologically or abnormally reduced levels of such lipids. These noveluses will be described in detail below.

In addition, the present invention relates to some novel compositions ofmatter being lipid mixtures, essentially mixtures of esters of LC-PUFAwith glycerol, which may be LC-PUFA mono-, di- and/or triglycerides,which may be derived from any suitable animal, plant, algae ormicroorganism source, with increased content of phytosterol orphytostanol esters, which can be used for lowering blood cholesterol andtriglycerides levels, as well as for the novel uses to be describedbelow.

Thus, the present invention relates to various therapeutic uses of theabove mixtures, as well as to some such novel mixtures. The novelmixtures of the invention also have particular advantages in deliveringrelatively high quantities of phytosterols and LC-PUFAs, and can be usedin specific dosage unit forms.

The mixtures used in the present invention consist essentially ofphytosterol and/or phytostanol ester(s) of LC-PUFAs in mixture withacylglycerol(s), which may be mono-, di- or triacylglycerols. Theglycerides are preferably mainly 1,3-diacylglycerols and triglycerides.The mixtures can also contain free sterols. The content of triglycerideesters of LC-PUFA in the mixtures of the invention is higher than thatof diglyceride esters of LC-PUFA. It is to be noted that the fraction ofglyceride esters of LC-PUFA (omega-3 fatty acids), may also containesters of fatty acids other than omega-3 fatty acids. Usually, theglyceride esters mixtures used by the present invention are of highconcentrations of DHA/EPA, and most of the LC-PUFA glyceride esters areof these acids. The same applies to the phytosterol ester fraction,which although predominantly comprises esters of LC-PUFA, may containesters of other fatty acids.

The mixture preferably comprises at least 10 wt % LC-PUFAacylglycerol(s), of which at least 5% are diacylglycerols, and at least5 wt % phytosterol and/or phytostanol ester(s) dissolved or dispersed insaid oil and/or fat. It is to be mentioned that throughout thisapplication, whenever referring to % of PUFA in the composition of theinvention or in fish oils, it is to be taken to mean % of LC-PUFA fromtotal fatty acids, and not weight percent from total weight ofcomposition.

The amount of diacylglycerol(s) contained in the mixtures may range from2 wt % to about 40 wt %, preferably from about 4 wt % to about 25 wt %and most preferably from about 6 wt % to about 22 wt %.

The amount of phytosterol and/or phytostanol ester(s) contained in theoil may range from 5 wt % to about 85 wt %, preferably from about 20 wt% to about 70 wt % and most preferably from about 25 wt % to about 65 wt%.

The diacylglycerol(s) consist substantially of 1,3-diacylglycerol(s)which mainly contain omega-3 unsaturated fatty acid residues. Thestructure of the diacylglycerol(s) depends on the particular oil or oilconcentrate used for mixing with the phytosterol ester(s) or forproducing the phytosterol esters. Preferably, fish oil concentratescontaining more than 30%, preferably more than 50%, more preferably60-80% LC-PUFA, particularly DHA and EPA, may be used. Other oils richin omega-3 fatty acids, preferably EPA and DHA, from another source,such as other animal, plants, algae or microorganisms are suitable.

The phytosterol and/or phytostanol ester(s) may be any phytosteroland/or phytostanol ester. Examples of such esters are stigmasteryl,sitosteryl and beta-sitosteryl esters of DHA and EPA.

The ratio between the glyceride esters and the sterol esters can varyfrom about 19:1 to 1:9, preferably from 9:1 to 1:9, more preferably from9:1 to 1:3, even more preferably from 3:1 to 1.5:1.

The ratio between the diacylglycerol(s) and the sterol esters can varyfrom about 1:40 to 1.5:1, preferably from 1:11 to 1:1.5, more preferablyfrom 1:9 to 1:3.

Thus, the principal candidates for inclusion as an ester attached to,e.g., sitosterol and campesterol, are the fish oil fatty acids EPA andDHA. It is proposed that esterification of plant sterols with EPA andDHA not only improves the efficacy of the plant sterols by moreaggressive exclusion of cholesterol from the micelle in the intestine,but also delivers to body cells fatty acids of the omega-3 class, whichare considered to possess several health benefits over and above theirfunction in lowering circulating triglyceride levels. Such beneficialeffects include improvement of immune function and risk reduction fordiseases including cancer, diabetes and arthritis. As will be discussedin detail, the multifunction mixtures of the invention confer thereforea portfolio of beneficial effects to health and well being.

The mixtures of the invention can be prepared by reacting fish oil withfree phytosterols using enzyme catalysis to affect the in situesterification of the phytosterols with fatty acids of the fish oil. Theenzyme can be a lipase enzyme, preferably immobilized and/or preferablycoated, as described in Applicant's WO00/56869. Specific procedures aredescribed in WO03/064444. The phytosterols can be of plant source,preferably soybean phytosterols or wood sterols. The sterols can beeither sterols or stanols (the terms phytosterol and phytostanols areused herein interchangeably). The fish oil can be of various grades andconcentrations of EPA and DHA. Preferably the fish oil is rich inDHA/EPA, preferably higher than 30%, preferably higher even than 50%.The fish oil can contain just EPA or just DHA or a mixture of thesefatty acids at different ratios. Preferably the fish oil contains higherlevels of DHA than EPA. The omega-3 fatty acids containing oil can alsobe any oil rich in these fatty acids, such as microbial or algae oilsrich in EPA and DHA. Other plant or animal sources of such oils are alsowithin the scope of the invention.

Generally, the immobilized enzyme is added to a stirred mixture of freesterols and fish oil at room temperature or at elevated temperature,preferably above 30° C., more preferably between 30 and 60° C. Thereaction mixture is stirred continuously for several hours. The reactionis followed by standard and appropriate analytical tools, such as HPLC,GC, TLC, NMR, MS, etc. Upon completion of the reaction, the reactionmixture is filtered to remove the catalyst. The filtrate can be used asthe active mixture. Preferably the filtrate is treated by standard oilprocessing techniques to remove free fatty acids, to maintainsatisfactory peroxide value, to improve color, etc. Treatment mayinclude distillation, steam deodorization, molecular distillation,bleaching, etc. Additives, such as antioxidants can be added to thefinal mixture to maintain its stability and quality.

Alternatively the active mixture of the invention, i.e. theOmega-3-esters mixture, can be prepared chemically by catalyzing theabove mentioned reaction with metal alkoxides, preferably with sodiummethoxide. This basic catalyst also confers transesterification thatyields phytosterol esters where the fatty acids of said esters are fromthe fish oil. In this route, the reaction, upon its completion, isneutralized by standard techniques, preferably by washings with anacidic aqueous solution. These washings can be further followed bywashings with water to maintain neutral pH levels. The active mixture isthen further processed as above.

In both procedures the level of phytosterol esters in the final mixturecan be controlled by the amount or percentage of free sterols added tothe fish oil. Levels of up to 70% can be easily obtained by thesemethods.

These in situ procedures yield as a by-product diglycerides, which arealso ingredients of said mixture. The level of diglycerides is alsocontrolled by the level of sterols used since the transesterificationreaction yields stoichiometric amounts of partial glycerides.

The active ingredient can also be prepared by esterifying free sterolswith a source of omega-3 fatty acids, preferably DHA and EPA. Thissource can be alkyl esters of omega-3 fatty acids, preferably ethyl ormethyl esters. The reaction is carried out enzymatically or chemically,as described above. The resulting phytosterol esters are purified fromthe alkyl esters by conventional methods and then mixed with fish oilsor other DHA/EPA-rich oils, which preferably may contain partialglycerides, particularly diglycerides.

Another aspect of the invention relates to a novel use of a combinationcomprising diacylglycerols and phytosterol and/or phytostanol ester(s)as an agent capable of reducing blood levels of both cholesterol andtriglycerides and/or for lowering serum, serum LDL and macrophageoxidation levels, inhibiting the formation of foam cells and/orpreventing the deleterious effects generated by lipid-induced oxidativestress.

The combination described in Examples 1-3 consists essentially ofphytosterol and/or phytostanol ester(s) and diacylglycerol(s), mainly1,3-diacylglycerols, preferably dispersed in an edible oil and/or fat,wherein the ratio of PSE to DAG is at least 1. More particularly, thecombination comprises at least 1 wt % diacylglycerol(s) and at least 1wt % phytosterol and/or phytostanol ester(s) dissolved or dispersed insaid oil and/or fat.

The amount of diacylglycerol(s) contained in the oil and or fat mayrange from 1 wt % to about 99 wt %, preferably from about 7 wt % toabout 48 wt % and most preferably from about 10 wt % to about 22 wt %.

The amount of phytosterol and/or phytostanol ester(s) contained in theoil may range from from about 1 to about 99 wt %, preferably from about5 to about 70 wt %, more particularly from about 7 to about 60 wt %,specifically from about 10 to about 60 wt %, more particularly fromabout 7 to about 35 wt % and more specifically from about 20 to about 35wt %.

The diacylglycerol(s) consist substantially of 1,3-diacylglycerol(s).The fatty acid profile of the diacylglycerol(s) depends on theparticular oil and/or fat used for producing the PSE and DAGcombination. For example, when olive oil is used, the diacylglycerolsmainly consists of 1,3-dioleyl glycerol. Generally speaking, fatty acidmoieties of the DAG may include, for example, caproic, caprylic,myristic, oleic, palmitic, palmitoleic, stearic, linoleic, linolenic,eicosanoic fatty acids and docosanoic fatty acids, but may contain otherfatty acyls.

The phytosterol and/or phytostanol moiety of the corresponding esters ofthe combination of the invention may be any phytosterol or phytostanolderivative. These phytosterols and/or phytostanols can be from soy,wood, algae, and other plants, as well as from animal or microbialsources that contain phytosterols and/or phytostanols. Examples ofphytosterols and/or phytostanols include but are not limited tobeta-sitosterol, stigmasterol, campesterol, brassicasterol,beta-sitostanol, campestanol, and stigmastanol.

With regards to the fatty acid moiety of the fatty acid phytosteroland/or phytostanol esters of the PSE+DSG combination, it may include anyfatty acid and more preferably different fatty acids characterizing thesource of the fatty acids. Such source may be a natural, synthetic, orfractionated oil or fat used in the interesterification of saidphytosterols and/or phytostanols. In other examples the source of thefatty acids may be any other esters of fatty acids, such as alkyl estersof fatty acids, preferably ethyl or methyl esters, or free fatty acids.The fatty acids may include, but are not limited to, caproic, caprylic,myristic, oleic, palmitic, palmitoleic, stearic, linoleic, linolenic,eicosanoic fatty acids and docosanoic fatty acids. It should beemphasized that in a preferred embodiment the combination of theinvention is produced by the in situ esterification, chemical orenzymatic, of a preferred oil and/or fat with a source of phytosterolsand/or phytostanols, simultaneously yielding diglycerides (DAG) and thusobtaining the combination of the invention in which the fatty acidprofile of the DAG and the fatty acids of the sterol or stanol esterscorresponds to the fatty acid profile of the preferred oil and/or fatused for the interesterification. In this process, the resultingcombination is dissolved or dispersed in the source oil and/or fat andcan optionally be further purified. The combination of the invention mayalso be obtained by separately producing phytosterol and/or phytostanolesters with a preferred source of fatty acids and combining saidphytosterol and/or phytostanol esters with DAG. The latter can becommercially available or produced by conventional chemical or enzymaticprocesses, including hydrolysis, alcoholysis, transesterifications orinteresterification processes. The fatty acid profile of the DAG of thecombination of the invention corresponds to the fatty acid profile ofthe source oil and/or fat used in their preparation.

The weight ratio of phytosterol and/or phytostanol ester(s) todiacylglycerol(s) in the combination or composition of the invention isat least 1:1. This weight ratio may be from about 15:1 to about 1:1,preferably about 10:1 to about 1:1, more preferably 5:1 to 1:1 andparticularly about 2:1. As used in the context of this application,ratios between various constituents are to be taken as weight ratios,unless specified otherwise.

The oil comprised in the PSE+DAG composition of the invention may be anyedible oil, including, but not limited to olive oil, soybean oil,sunflower oil, safflower oil, canola oil, sesame oil, palm oil, avocadooil or fish oil. Preferably the oil is at least one of olive oil, canolaoil or fish oil. The fat contained in the composition of the inventionmay be any suitable fat, such as, e.g., butter fat, anhydrous milk fat,cocoa butter, as well as animal fat such as lard or a fish oilconcentrate.

The diacylglycerol(s) may be obtained by any conventional enzymatic ornon-enzymatic procedure. Preferably, they are obtained byinter-esterification reaction between phytosterol(s) and triglyceride(s)present in the oil and/or fat. The phytosterol and/or phytostanolester(s) may be obtained by any conventional enzymatic or non-enzymaticprocedure. Preferably, these constituents are obtained byinteresterification reaction between phytosterol(s) and/or phytostanoland triglyceride(s) present in an edible oil or fat. A process forobtaining the combinations used by the present invention is described indetail in WO03/064444, fully incorporated herein by reference.

As shown in the following examples, a significant effect of the testedcompositions in preventing and/or reducing serum ox-LDL, as well asmacrophage oxidation was obtained. Thus, in addition to having an effectin reduction of blood LDL-cholesterol and triglycerides levels, the saidcombination, and compositions comprising the same, exhibit serum LDL andmacrophage anti-oxidative properties. The examples further show thatpreparation of PSE+DAG in olive oil, canola oil and/or fish oilexhibited significant anti-oxidative properties, inhibiting theformation of foam cells, and/or preventing the deleterious effectsgenerated by lipid-induced oxidative stress.

Specifically, FIGS. 5, 7, 8 and 9 demonstrate how the different PSE+DAGpreparations lowered the following oxidative stress parameters: serumoxidative stress, ox-LDL uptake by peritoneal macrophages, macrophageoxidative status, and PMA-induced superoxide anions release frommacrophages.

PON1 activity in the serum was also measured. PON1 is an HDL-associatedesterase that can eliminate ox-LDL. Interestingly, the results of FIG. 6show that while canola oil reduces PON1 activity, the PSE+DAGcombinations of the invention were able to maintain PON1 higher levels,suggesting that the combination of the present invention may protectPON1 activity in a pro-atherosclerotic environment.

The combinations/compositions of PSE+DAG were most effective in reducingtotal cholesterol (see FIG. 10) and LDL cholesterol plasma levels (seeFIG. 11) as well as fasting triglycerides levels (see text below andExample 3).

In addition, subjects who consumed the combination/composition of theinvention showed a pronounced reduction of apo B values (FIG. 12).Consequently, there was also a positive change, i.e. decrease, inapoB/apoA ratio. Apo B, apo A-I and the apo B/apo A-I ratio have beenreported as efficient predictors of cardiovascular events than LDL-C andas may be seen from FIG. 13, in addition to the aforementioned reductionin apo B levels, introducing PSE+DAG to the base MUFA-enriched dietconsumed by the volunteers resulted in significantly reduced levels ofapoB/apoA ratios.

Lipoprotein(a) (Lp(a)) is an LDL-like particle to which apo A isattached through a disulfide bond to apo B. Increased plasma levels ofLp(a) are an independent predictor of the presence of angiographicallydocumented and clinical CAD, particularly in patients withhypercholesterolemia [Danesh J., et al. (2000) Circulation102:1082-1085]. In the present human study, hypercholesterolemicvolunteers fed with control diet presented significantly elevated levelsof Lp(a) (FIG. 14).

The present human study also demonstrates a pronounced and significantreduction in plasma oxidative stress levels (FIG. 15).

In Example 4, the inventors have now established various activities ofthe Omega-3-esters mixture of the invention (Preparation A).

Cholesterol and Lipoproteins

As shown in FIG. 16, a substantial decrease in total cholesterolconcentrations was observed following the fish oil treatment, whilecontrol diet consumption induced a significant though smallerhypocholesterolemic effect. In terms of reduction of absolutecholesterol concentrations, the volunteers that consumed these dietsreduced 21 mg/dL and 13 mg/dL; from initial average level of 241 mg/dLand 240 mg/dL to a final average levels of 220 mg/dL and 228 mg/dL,respectively. With respect to the American Heart Association (AHA)recommendations, as were expressed in the Third Report of the Expertpanel on Detection, Evaluation, and Treatment of High Blood Cholesterolin Adults (Adult Treatment Panel III or ATP III) released in 2001, thedesirable total cholesterol levels in healthy individuals should bebelow 200 mg/dL. Remarkably, treatment with Preparation A of theinvention resulted in the largest change in total cholesterol comparedto diets containing no supplemented plant sterols (−12.52% versus −8.45%and −4.40%; P-Value=0.039). Importantly, the decreases in totalcholesterol levels induced by Preparation A could be regarded asreducing 72% of the total cholesterol levels towards the recommendedATPIII guidelines (from 243 mg/dL to 212 mg/dL).

These effects are interesting from a physiological point of view and inaccordance with the existing data on the hypocholesterolemic effect ofplant sterols. Indeed, plant sterols, at a dose ranging from 1.5 to 2.5g/day, have been reported to decrease total cholesterolemia, due mainlyto a 10% decrease in LDL cholesterol levels [Katan et al. (2003) MayoClin. Proc.; 78:965-78]. Moreover, in a recent review of fish oilsupplementation effect on lipids levels in diabetic type 2 patients[Montori et al. (2000) Diabetes Care; 23:1407-1415] a fairly minoreffect was demonstrated for altering total cholesterol levels (+0.4mg/dL [95% CI −5.0 to +5.8]). Interestingly, when comparing patientswith plasma cholesterol levels above 265 mg/dL at entrance to the study(n=6) that consumed fish oil to Preparation A, a significant beneficialeffect of the Preparation A diet was observed on absolute or % of change(P=0.04) in total cholesterol levels. Thus, the mixtures of theinvention, and particularly Preparation A, are useful in loweringcholesterol levels.

As shown in FIG. 17, this hypocholesterolemic effect of Preparation Awas more prominent, as expected, in the reduction of LDL cholesterolplasma levels, while fish oil consumption resulted in an elevation ofLDL cholesterol concentrations, compared to the reduction in LDLcholesterol obtained by the control diet. Fish oil has been previouslyshown to induce an increase in LDL cholesterol concentrations [Harris etal. (1991) J Am Coll Nutr.; 10(3):220-7]. This increase in LDLcholesterol levels may be associated with larger LDL molecules of lowerdensity. In the present study, Preparation A attenuated the increasingeffect of fish oil on LDL cholesterol (from 158 mg/dL to 144 mg/dL;P-Value=0.006). Indeed, the changes in the LDL-cholesterolconcentrations after supplementation with Preparation A werestatistically different from those observed after fish oilsupplementation (−8.04% versus −0.93%, respectively; P<0.05). However,the multiple comparison of Preparation A and fish oil with the controldiet resulted in a fairly mild tendency (P=0.322), most probably due tothe variation within the treatments groups. Nonetheless, a pairedanalysis of the effect of Preparation A on LDL-cholesterol compared tothe control diet had demonstrated a marked tendency (8.04% versus 4.13%;P-value=0.15). In terms of ATPIII recommendations for LDL-cholesterol,which are set, for subjects who do not have coronary heart disease ordiabetes, to be less than 130 mg/dL, treatment with Preparation Aresulted in 49% reduction of the amounts of LDL cholesterol required toreach this goal, while the control diet and the fish oil treatmentinduced 31% and 15% reduction, respectively. The mixtures of theinvention are thus not only useful in lowering total cholesterol, butadvantageously reduce levels of LDL cholesterol.

Moreover, type 2 diabetes is frequently associated with dyslipidemia.Recently, it was found that small dense LDL particles are often presentin type 2 diabetes [Sniderman et al. (2001) Ann. Int. Med. 135:447-459].Although abnormal LDL composition has gained notice, much less attentionhas been paid to the issue of LDL particle number. This gap in knowledgeis important because it was suggested that in diabetics there areincreased numbers of small dense LDL particles in association withhypertriglycerides. This combination constitutes of hypertriglyceridemicand hyper-apoB, is one of the most common dyslipidemias associated withpremature vascular disease.

As shown in FIG. 18, the mixture of the invention had a more pronouncedreduction effect on endpoint apolipoprotein B₁₀₀ (apoB) concentrations(−12.52%), compared to the effect obtained by un-reacted fish oil(−8.84%) or control diet (−2.27%). The compositions of the invention,where the preferred embodiment is preparation A, may thus be useful inreducing apoB concentration. The effect of fish oil on apoBconcentrations is still unclear. Previous studies have shown decreased[Sanchez-Muniz et al. (1999) Eur J Nutr.; 38(1):20-7) or increased apoBlevels after fish oil supplementation [Calabresi et al. (2004)Metabolism; 53(2):153-8]. The present results suggest that the omega-3esters mixture of the invention may decrease the number of circulatingatherogenic apoB-containing lipoproteins after one month ofsupplementation as a combined effect of the plant sterols and fish oil.The mixture could be particularly important to diabetics, as apoB₁₀₀levels were shown to be associated with the metabolic syndrome and,possibly, to insulin-insensitivity in Type 2 diabetes patients [Relimpioet al. (2002) Diabetes Res. Clin. Practice; 57(3):199-207].

As shown in FIG. 19, when the omega-3 esters mixture of the invention(Preparation A) was compared to the fish oil and control diets,significant differences were observed between the effects of treatmenton apoA/apoB ratios absolute changes (0.09, −0.01 and 0.02,respectively; P=0.039) and % change (8.45%, 0.35% and 2.20%,respectively; P=0.09). This effect could be attributed to the relativemaintanance of apoA concentrations while generating a significantdecrease in apoB levels as a result of supplementation with mixtures ofomega-3 esters. Interestingly, a paired analysis between the mixture ofomega-3 esters and the fish oil consumption had suggested a distinctdifference between the effects of these supplements (P-Value=0.005). ApoB, apo A-I and the apo B/apo A-I ratio have been reported as betterpredictors of cardiovascular events than LDL-C and they even retaintheir predictive power in patients receiving lipid-modifying therapy[Walldius et al. (2004) J Intern Med.; 255(2):188-205].

When fish oil was trans-esterified with plant sterols (Preparation A)and compared to the consumption of the control diet, a significantlydecreased total/HDL cholesterol ratio was demonstrated (compared to fishoil and control diet; see FIG. 20). These results suggest that thetrans-esterification with plant sterols not only counteracts theincrease in total/HDL cholesterol ratio, but may confer to omega-3LC-PUFA a beneficial impact on cholesterol profile, which is notobserved with un-reacted fish oil. This reduction in totalcholesterol/HDL cholesterol ratio with the omega-3 esters mixture of theinvention may be of particular interest for patients presenting bothelevated triglyceride levels and total/HDL cholesterol ratios. Theimportance of this parameter was demonstrated in the Quebeccardiovascular study [Lemieux et al. (2001) Arch Intern Med.;161(22):2685-92], in which it was concluded that the variation in thetotal/HDL cholesterol ratio may be associated with more substantialalterations in metabolic indices predictive of ischemic heart diseaserisk (IHD) and related to the insulin resistance syndrome than variationin the LDL-C/HDL-C ratio. Given the beneficial impact of the mixtures ofomega-3 esters on reducing the total/HDL cholesterol ratio and apoBconcentrations, the omega-3 esters mixture of the invention seems to bea promising alternative to fish oil treatment in hypertriglyceridemicpatients with elevated cholesterol levels.

As demonstrated in the present study, feeding hypercholesterolemicsubjects for a limited period with either control diet or fish oil hadno effect on the HDL₂ subfraction levels. However, supplementation withthe omega-3 esters mixture of the invention (Preparation A) resulted ina marked elevation in HDL₂ levels, which was shown to be significantlydifferent than the control diet or fish oil treatment effects (FIG.21A). The HDL₃ levels were reduced similarly by either the fish oil, butwere not affected by the control diet consumption, as shown in FIG. 21B.Thus, the omega-3 esters mixture of the invention (Preparation A) wasshown to have a potent effect on HDL subfractions. This is an importantfeature of the invention, which provides for use of the mixtures in theelevation of HDL₂, whilst reducing levels of HDL₃. The protective roleof HDL against the development of IHD is well accepted; severalprospective studies have confirmed the early observations of Barr andcolleagues [Barr et al. (1951) Am J Med.; 11:480-493] who firstsuggested more than 50 years ago that individuals with elevated plasmaconcentrations of HDL cholesterol were at lower risk for IHD.

Generally, the HDL consists of a heterogeneous group of particlesdefined either by size or by apolipoprotein content. Subfractions of HDLappear to have distinct but interrelated metabolic functions, includingfacilitation of cholesteryl ester transfer to LDL and VLDL, modulationof triglyceride-rich particle catabolism, and, possibly, removal ofcholesterol from peripheral tissues. Like HDL cholesterol, HDLsubfractions are widely affected by a variety of factors. HDLsubfractions augmented levels are also markers for epidemiologic riskfor coronary artery disease. Specifically, results from both the KuopioIHD Risk Factor Study and the aforementioned Quebec study have suggestedthat the cardioprotective effect of elevated HDL cholesterol levels maybe attributed to the HDL₂ subfraction. Because they provide informationabout the physiologic processes of cholesterol metabolism, HDLsubfractions are emerging as an increasingly important tool in the studyof the relationship between lipids and CVD. Indeed, it was shown thatobesity [Laakso et al. (1990) Metabolism; 39:117-22] and insulinresistance [Tilly-Kiesi et al. (1996) J Lipid Res.; 37:1569-78] areassociated with low total HDL and HDL₂ and a higher proportion of HDL₃.The reductions in HDL associated with type 2 diabetes and insulinresistance are multi-factorial, but a major factor appears to beincreased transfer of cholesterol from HDL to triglyceride richlipoproteins, with reciprocal transfer of triglyceride to HDL.Triglyceride-rich HDL particles are hydrolyzed by hepatic lipase and, asa result, are rapidly catabolized and cleared from plasma [Hopkins andBarter (1986) J Lipid Res.; 27:1265-77]. Krauss [Krauss (2004) DiabetesCare; 27:1496-1504] had indicated that typically the reduced HDL levelsin plasma of patients with type 2 diabetes are manifested as reductionsin the HDL_(2b) subspecies and relative or absolute increases in smallerdenser HDL_(3b) and HDL_(3c). In normolipidemic human subjects there arejust a few indications on fish oil consumption resulting with HDLsubfraction alterations. However, dyslipidemic diabetic subjects werereported to benefit of fish oil supplementation in a specific HDL₂rising levels while HDL₃ shown tendency to decline, suggesting a shiftin the HDL profile in the direction of larger and less dense particles[Dunstan et al. (1997) Diabetes Care; 20:913-921; Fashing et al. (1996)Horm Metab Res.; 28:230-6; Lou et al. (1998) Diabetes Care; 21:717-24].Nevertheless, none of these studies in dyslipidemic patients haddemonstrated comparable alterations as described in FIG. 21.

Triglycerides and Fatty Acids

The mixtures of the invention also have beneficial effects ontriglycerides and fatty acid level. As shown in FIG. 22, un-reacted fishoil decreased plasma fasting triglycerides by 37.1%, while thecomparable effect of the omega-3 esters mixture of the invention(Preparation A) was 42.9%. Comparing the effects of these supplements onfasting triglycerides levels to the rather mild effect of the controldiet resulted in a statistically significant difference between theconsumption of fish oil to olive oil. A recent meta-analysis, however,suggests that a 8.8 mg/dL increase in triglyceride concentration isassociated with an increase in the frequency of CVD in both men andwomen [Austin et al. (1998) Am. J. Cardiol.; 81:7B]. Indeed, the AdultTreatment Panel III identifies elevated serum TG concentrations as anindependent risk factor [ATP III]. Clinical studies of fish oilsupplementation with a parallel or cross-over study design have reporteda dose-dependent triglyceride lowering effect for omega-3 fatty acids inboth normolipidemic [Blonk et al. (1990) Am. J. Clin. Nutr. 52:120] andhyperlipidemic subjects [Davidson et al. (1997) J. Am. Coll. Nutr.;16:236]. As shown in the results presented herein, consumption of theomega-3 esters mixture of the invention (Preparation A) generated atriglyceride reduction, as depicted in FIG. 22.

Similar effects, but with a higher tendency, were observed onpostprandial triglyceride levels (FIG. 23), where the lowest plasmatriglyceride concentrations after consumption of a meal were detected inthe subjects fed with the omega-3 esters mixture of the inventioncompared with fish oil and control diet supplements. Interestingly, thepaired analysis between the un-reacted fish oil and the omega-3 estersmixture of the invention (Preparation A) demonstrated a notable tendencybetween these two supplements, though the levels of the EPA and DHA wereidentical. It has been suggested that postprandial triglycerideconcentrations are a better marker as compared to the fasting levels oftriglyceride for determination of risk factor for CHD. This analysis mayshow a state of fat tolerance which cannot be detected by fastingtriglyceride measurements [Karpe (1999) Intern. Med., 246:341].Moreover, Hansen and colleagues [Hansen et al. (1998) Lipids; 33:131-8]reported that healthy volunteers fed by either EPA or DHA shown that thepostprandial triglyceridemia was suppressed by 19 and 49% afterprolonged intake of EPA and DHA, respectively, indicating that prolongedintake of DHA is equivalent to or even more efficient than that of EPAin lowering postprandial triglyceridemia. Taking both the fasting andthe postprandial triglycerides levels alterations as presented in FIGS.22 and 23 suggest that the hypotriglyceridemic effect of fish oil fattyacids is not only retained when they are esterified to plant sterols butgenerates a more prominent impact on circulating lipids.

Plasma fatty acid analysis clearly indicates that following the omega-3esters mixture of the invention (Preparation A) consumption, the levelof DHA is significantly higher than that present in a subject whichconsumed equivalent amounts of fish oil. These levels in both omega-3based treatments are, of course, significantly elevated compared to thecontrol diet. As the administered levels of DHA were identical in bothfish oil based treatments, these notable observations could imply asuperior bioavailability of DHA esterified to plant sterols comparing totriglycerides. This suggested mode of action is further backed by theanalysis of blood plant sterols levels. The increase in campesterolconcentrations was more pronounced than the increase in beta-sitosterollevels, reflecting a higher absorption of campesterol. However, theomega-3 esters mixture of the invention increased sitosterolconcentrations more than non-esterified sterols, suggesting a higherbioavailability of sitosterol when supplied as fish oil esters than asunesterified sterols. Diabetes was shown to impair essential fatty acidmetabolism by decreasing activities of Δ6- and Δ5-desaturases, enzymesthat convert dietary linoleic acid and γ-linolenic acid to long-chainpolyunsaturated fatty acids (LC-PUFA), including γ-linolenic acid,arachidonic acid (AA), EPA, and DHA [Horrobin (1998) ProstaglandinsLeukot Essent Fatty Acids; 31:181-97]. As a result, AA and DHA levelsare reduced in membrane phospholipids of several tissues, includingerythrocyte and sciatic nerve, in patients with type 1 diabetes and indiabetic animals [Ruiz-Gutierrez et al. (1993) Diabetologia; 36:850-6].It was demonstrated recently that dietary supplementation with fish oil,containing EPA and DHA [Gerbi et al. (1999) J. Nutr.; 129:207-13],partially prevented the diabetes-induced decrease in nerve conductionvelocity, a physiological marker of diabetic neuropathy. This wasfurther correlated in a recent report [Coste et al. (2003) Diabetes;52(10):2578-85] that presented evidence for a marked neuro-protectiveeffect of DHA on diabetic neuropathy.

Inflammatory Markers

Furthermore, the mixtures of the invention may have a beneficiary effectin the control of inflammatory process. C-reactive protein (CRP) is aphylogenetically highly conserved plasma protein that participates inthe systemic response to inflammation. Its plasma concentrationincreases during inflammatory states, a characteristic that has longbeen employed for clinical purposes. Recent studies have demonstratedthat intensive treatment like statin produced greater reductions in bothLDL cholesterol and CRP, suggesting a relationship between these twobiomarkers and CVD progression [Nissen et al. (2005) N. Eng. J. Med.;352:29-38]. Indeed, several studies have demonstrated that increased CRPconcentrations are predictive of vascular events even among thosewithout hyperlipidemia. In fact, CRP monitoring, among other analyses,was recommended by Naghavi and colleagues review on vulnerable plaques[Naghavi et al. (2003) Circulation; 108:1664-72] as a key parameter.Accordingly, Rifai and Ridker have proposed an algorithm forcardiovascular risk prediction using both CRP and Total/HDL-Cholesterolratio [Rifai and Ridker (2001) Clin. Chem.; 47:28-30]. In the presentstudy the comparison of short-term consumption of control diet with orwithout fish oil or the omega-3 esters mixture of the invention(Preparation A) exhibited only a mild tendency towards altered effects(FIG. 24). However, in a paired analysis it was distinctly shown thatCRP levels following the omega-3 esters mixture of the invention(Preparation A) treatment were significantly reduced, compared withcontrol diet (P-value=0.058) and fish oil (P-value=0.042). Furthercorrelations between the total to HDL-cholesterol ratio and CRP-levelshad remarkably indicated a reduced relative risk of CVD eventprospective in the following ten years for the subjects who consumed theomega-3 esters mixture of the invention (FIG. 25).

In parallel to the aforementioned suggestion that elevated plants sterollevels could serve as reinforcement to the superior bioavailability offish oil attached to the plant sterols, it could play a role in adifferent biological process. The action of plant sterols as anticancerdietary components has been recently extensively reviewed [Awad et al.(2000) J. Nutr.; 130:2127-30]. Plant sterols can suppress tumor cellgrowth (LNCaP and HT-29) [Awad et al. (2000) Nutr Cancer; 36:74-8].Compared to cholesterol, beta-sitosterol caused a 24% decrease in cellgrowth and a 4-fold increase in apoptosis. Finding in SCID micexenografted with the human breast cancer cell line implied thepossibility that plant sterols may retard the growth and spread ofbreast cancer cells [Awad et al. (2000) Anticancer Res.; 20:821-4]. Inaddition to retarding the growth of breast cancer cells by plantsterols, there is some evidence that plant sterols can affect thedevelopment of prostate cancer [Wilt et al. (2000) Cochrane DatabaseSyst 2]. In a meta-analysis, 519 men were studied in 4 randomized,placebo-controlled, double-blind trials. Beta-sitosterol improvedurinary symptom scores and flow measures, suggesting that non-glucosidicforms of beta-sitosterol improve urinary symptoms and flow measures.

In the present study there was indeed a mild tendency of prostatespecific antigen (PSA) reduction (−5.2%) comparing to the control diet(0.22%). The low statistical significant could be attributed to therelative low levels of PSA detected in the subjects and the highvariability.

TNF-α and IL6

While the n-6 polyunsaturated fatty acid (PUFA), arachidonic acid (AA),is a precursor of prostaglandins, leukotrienes and related compoundsthat have important roles as mediators and regulators of inflammation,consuming increased amounts of long chain n-3 PUFA (found in oily fishand fish oils) results in a partial replacement of the AA in cellmembranes by DHA and EPA. This leads to decreased production ofAA-derived mediators. This alone is a potentially beneficialanti-inflammatory effect of n-3 fatty acids. Moreover, animal studieshave provided a great deal of evidence that feeding plant or fish oilsrich in n-3 PUFAs does alter the ex vivo production of tumor necrosisfactor (TNF), interleukin 1 (IL-1), IL-6 and IL-2, but manycontradictory observations have been made; it is most likely that thediscrepancies in the literature result from differences in the celltypes and experimental protocols used. Human studies provide moreconsistent data; several studies have shown that supplementation of thediet of healthy volunteers results in reduced ex vivo production ofIL-1, IL-6, TNF and IL-2 by peripheral blood mononuclear cells [Calder(1997) Ann Nutr Metab.;41(4):203-34]. Recently, studies conducted inobese [Jellema et al. (2004) Eur J Clin Invest.; 34(11):766-73] anddiabetic patients [Mori et al. (2003) Free Radic Biol Med.;35(7):772-81] administrating moderate to high dosage of purified EPAand/or DHA failed to demonstrate a comparable alterations in thesesubjects blood inflammation markers levels. In the present study it wasshown that as expected, the control diet had no effect on TNF-α and IL-6levels, while similar feeding period with fish oil resulted withaugmented levels of these markers. However, the omega-3 esters mixtureof the invention (Preparation A) treatment had generated the largesteffect on these inflammation markers levels.

In a further aspect, the invention relates to unique dosage unit form,for the delivery of an effective dose, preferably the RDI (recommendeddaily intake), of both phytosterols and DHA/EPA.

Different active dietary ingredients, such as vitamins, have arecommended daily allowance (RDA), provided by different healthauthorities, such as the US Food and Drug Administration (FDA). The RDIof sterols, according to the US FDA is 0.8 g/day. This RDI is supposedto provide the cholesterol reduction effects attributed to phytosterols.The International Society for the Study of Fatty Acids and Lipids hadrecommended in April 1999 following the “Workshop on the essentiality ofand recommended dietary intakes for omega-6 and omega-3 fatty acids” anadequate intake of 650 mg of EPA+DHA per day in connection tocardio-protective effects. Since both phytosterols and omega-3 fattyacids such as DHA and EPA are linked to heart health it may be desirableto create mixtures of these two ingredients. In order to create ahomogenous blend of these two ingredients, the use of the oil-solublephytosterol-esters is necessary. When attempting to prepare a mixture ofcommercial phytosterol-esters and EPA/DHA omega-3 fatty acids one may berequired to consume high dosages per day, for example of up to 4-5 g ofmixture.

The RDI of plant sterol esters is 1.4 g/day, correlated to 0.8 g/day ofthe free phytosterols. Relatively pure (90-95% wt) plant sterol estersare commercially available. The most common source of DHA and EPA isfish oils, of which the most popular are those containing about 25 g/100g of EPA/DHA, usually in a ratio of 18:12. Thus, by using these twosources of phytosterols and EPA/DHA, in order to achieve the recommendedlevels described above, one would need to consume 4.2 g per day. Thisamount, provided in softgel capsules, taking into account the limitationof capsule size, may be divided to about 4-5 capsules. Such large numberof capsules per day is tedious to the health-conscious end consumer.Even when using more concentrated fish oil, such as containing about 55%wt of EPA/DHA, one would still need to consume about 2.75 g of themixture. This again results in 3 large capsules per day. However, marketresearch has shown that consumers prefer to consume dietary supplementsor pharmaceutical preparations by a maximum of 2 unit forms per day.

The present invention provides a solution to this problem by introducinga pharmaceutical dosage form of esters of LC-PUFA with phytosterolsand/or phytostanols, wherein said RDI of sterols is from about 0.4 g toabout 1.0 g, preferably from about 0.6 g to about 0.8 g. Said RDI ofLC-PUFA, specifically DHA and EPA is from about 0.3 g to about 0.8 g,preferably from about 0.5 g to about 0.65 g.

Since in the omega-3 esters mixture of the invention, and particularlyPreparation A, the phytosterol esters fraction also contains EPA and DHAfatty acids, the mixture of the invention can deliver the RDIs mentionedabove in a total amount of about 2 g per day. This amount can be easilydelivered by two (2), user friendly, capsules per day. This mixturecontains about 1.5-1.6 g of phytosterol esters, of which about 60% areesterified to EPA/DHA. Hence, the amount of phytosterol esterscorresponds to the RDA 0.8 g/day of phytosterols and 420 to 480 mg ofEPA/DHA. It is to be noted that phytosterol esters of LC-PUFAs weighmore than vegetable-oil derived fatty acids esterified to plant sterol,since LC-PUFAs are characterized by a higher molecular weight comparedto vegetable-oil derived fatty acids, usually containing only 16-18carbons. By mixing the above amount of LC-PUFA phytosterol esters withabout 400 mg of LC-PUFA rich oil, such as fish oil, containing about 60%EPA/DHA, one can easily obtain 660-720 mg of EPA/DHA in the finalmixture. Thus, this mixture contains both the recommended levels ofomega-3 LC-PUFAs and phytosterols in a total amount of up to 2 g, easilyprovided by 2 capsules. It is to be further noted that the omega-3mixture of the invention has omega-3 fatty acid moieties on both thephytosterol and the glyceridic fractions, thus being inherently capableof providing high quantities of phytosterol. This is a further advantageof the mixtures of the invention, particularly Preparation A.

Alternatively, the omega-3-esters mixtures may be the active ingredientof a pharmaceutical or nutraceutical composition for reducing bloodlevels of both cholesterol and triglycerides and/or for loweringcirculating small-dense LDL particles. In particular, these compositionsmay be used for reducing blood levels of HDL₃ whilst elevating levels ofHDL₂ lipid sub-fractions, and maintaining a high ratio of HDL/LDL. Thus,use of the mixtures and compositions of the invention may beadvantageous in reducing the relative risk of coronary events, in bothmales and females. In addition, the mixtures and compositions of theinvention may be useful in reducing risk of developing IHD,atherosclerosis and hypertension. A further important use is in theprevention and treatment of the metabolic syndrome (Syndrome X) andrelated conditions such as insulin resistance. Another use of themixtures and compositions of the present invention is in the treatmentof cancer.

In addition, pharmaceutical compositions comprising the PSE+DAGcombination as the active or auxiliary ingredient may be used forreducing blood levels of both cholesterol and triglycerides and/or forlowering oxidation levels of serum, serum LDL and macrophage oxidation,inhibiting the formation of foam cells and/or preventing the deleteriouseffects generated by lipid-induced oxidative stress.

The dosage of the PSE+DAG combination of the invention may depend uponthe condition to be treated, the patient's age, sex and bodyweight, andwill be determined by the attending physician or dietician. A preferreddosage for an adult may be from about 1.5 to about 6 g of PSE+DAG in oildispersion (in accordance with the invention) per day, preferably 2-5 g,which shall comprise approximately 1300 mg of PSEs and up to 800 mg ofDAGs.

Other formulations which may be in unit dosage forms may be as follows:

-   -   A combination of PSE and DAG dissolved in canola oil where the        fatty acids of the sterol esters and the DAG resemble the fatty        acid profile of canola oil, comprising including 31.5% wt PSE        and 10% wt DAG. Each 4.5 g of this combination will contain at        least 1300-1400 mg PSE (RDA) and 450 mg of DAG.    -   A combination of PSE and DAG dissolved in soybean oil, where the        fatty acids of the sterol esters and the DAG resemble the fatty        acid profile of soybean oil, comprising 31% wt PSE and 15.9% wt        DAG. Each 4.5 g of this formulation will contain at least        1300-1400 mg PSE (RDA) and 700 mg of DAG.    -   A combination of PSE and DAG dissolved in canola oil comprising        61% wt PSE and 10% wt DAG. Each 2 g will contain at least        1300-1400 mg PSE (RDA) and 200 mg of DAG.    -   A combination of PSE and DAG dissolved in fish oil where the        fatty acids of the sterol esters and the DAG resemble the fatty        acid profile of fish oil, comprising 61% wt PSE and 8.3% wt DAG.        Each 2 g will contain at least 1300-1400 mg PSE (RDA) and 150 mg        of DAG.    -   A combination of PSE and DAG dissolved in canola oil where the        fatty acids of the sterol esters and the DAG resemble the fatty        acid profile of canola oil, comprising 23% wt PSE and 8% wt DAG.        Each 6 g will contain at least 1300-1400 mg PSE (RDA) and 450 mg        of DAG.

Other PSE and DAG formulations as well as dilutions of all formulationsare within the scope of the invention.

The preparation of pharmaceutical compositions is well known in the art,see e.g., U.S. Pat. Nos. 5,736,519, 5,733,877, 5,554,378, 5,439,688,5,418,219, 5,354,900, 5,298,246, 5,164,372, 4,900,549, 4,755,383,4,639,435, 4,457,917, and 4,064,236. The omega-3-esters mixture used bythe present invention may be preferably mixed with an additive,excipient, carrier and/or diluent, and optionally, a preservative or thelike pharmacologically acceptable vehicles as known in the art, seee.g., the above US patents. Examples of excipients include glucose,mannitol, inositol, sucrose, lactose, fructose, starch, corn starch,microcrystalline cellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like.Optionally, a thickener may be added, such as a natural gum, a cellulosederivative, an acrylic or vinyl polymer, or the like.

The pharmaceutical composition is preferably provided in liquid, solidor semi-solid form. The liquid preparation is provided preferably as anoil suspension or microcapsule composition. A semi-solid composition isprovided preferably as oily gel or cream.

Tablets, hard tablets, caplets, capsules and particularly soft gelatincapsules, containing the omega-3-esters mixture of the invention may bepreferred, either as dietary supplements or as pharmaceutical dosageforms. In essence, any pharmaceutical dosage form suitable for oraladministration may be used for delivering the combination of theinvention.

The dosage of the active mixtures of the invention may depend upon thecondition to be treated, the patient's age, sex and bodyweight, andshall be determined by the attending physician or dietician. A preferreddosage for an adult may be from about 2 to about 6 g of omega-3 mixturesper day, e.g. 4 to about 6 g of Preparation A per day, preferably 5 g,which comprise approximately 1300 mg of PSEs and 800 mg of diglyceridicesters of LC-PUFA, preferably of DHA/EPA.

As described above, the present invention also provides mixtures whichare highly concentrated, and can deliver high doses of phytosterol andof DHA/EPA in dosage unit forms. Evidently when using the concentratedforms, lower doses may be used, depending mainly on the amount ofphytosterol esters.

The omega-3 mixtures, or the PSE+DAG combination or composition of theinvention may be used per se, as a food article. Such food article maybe any conventional food, and also a functional food or beverage.Alternatively, they may be an ingredient of a food article orsupplement, which may further optionally contain conventional additivesused in the food industry, such as preserving agents, colorants,flavoring agents, fragrances, antioxidative and hardening agents,vitamins, calcium, other minerals, trace elements, probiotic agents,isoflavones, caloric agents and the like.

In addition, the food supplements of the invention may be used in themanufacture of any one of functional foods, functional drinks or dietarysupplements. Said food supplement may be introduced into said food,drink or dietary supplement by admixing, adding or incorporating itduring manufacture thereof.

The terms dietary nutrient and food supplement may be used hereinexchangeably, and are to be taken to mean any edible supplements,particularly dietary supplements to edible products, preferably foodarticles, including functional foods and functional beverages.

As mentioned above, the mixtures and compositions of the invention maybe useful in reducing the risk of developing IHD, atherosclerosis andhypertension, as well as in the prevention and treatment of themetabolic syndrome (Syndrome X) and related conditions such as insulinresistance, and in the treatment of cancer. Thus, the present inventionalso refers to a method of treating and/or preventing any of the aboveconditions, by orally administering a therapeutically effective dosageof the active mixtures, food supplement or compositions of the inventionto a subject in need. Consequently, the method is also effective for thetreatment of cardiovascular disorders, coronary heart disease,atherosclerosis, as well as cardiovascular disorders induced ormanifested by other diseases such as diabetes mellitus, particularlyType II diabetes.

The present invention is particularly directed at a method of treatingand/or preventing conditions related to any one of high cholesterol andtriglycerides blood levels, serum oxidative stress, ox-LDL uptake bymacrophages, macrophage oxidative status, foam cells formation andlipid-induced oxidative stress, said method consisting of orallyadministering a therapeutically effective dosage of the combination orcomposition of the invention, in the form of a food supplement,nutraceutical or pharmaceutical composition to a subject in need.Consequently, the method is also effective for the treatment ofcardiovascular disorders, coronary heart disease, atherosclerosis, aswell as cardiovascular disorders induced or manifested by other diseasessuch as diabetes mellitus, particularly Type II diabetes.

Alternatively, such conditions are to be prevented by consumption of thedietary products in accordance with the invention.

Lastly, the present invention presents a method for improving health,consisting of administering a therapeutically effective dosage of thedietary nutrient comprising the omega-3-esters mixture of the invention,or the pharmaceutical or nutraceutical composition thereof to a subjectin need.

The present invention is defined by the claims, the contents of whichare to be read as included within the disclosure of the specification.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, process steps, and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and not intendedto be limiting since the scope of the present invention will be limitedonly by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The following Examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the intended scope ofthe invention.

EXAMPLES

Materials:

-   -   Olive oil: commercial extra virgin olive oil, manufactured by        Meshek Eger (Yokeneam HaMoshava, Israel).    -   Canola oil: commercial canola oil, manufactured by Shemen        Taasiot (Haifa, Israel).

Fish oil: commercial fish oil, manufactured by Pronova (Lysaker,Norway). TABLE 1 Composition of the PSE + DAG oils PSE + DAG in Oliveoil Oil PSE + Olive oil PSE + enriched with components % DAG in(Enzymotec FG DAG in phytosterol (w/w) Canola oil S7/1.75) Fish oilesters Phytosterol esters 26 28.5 22.12 18 Monoglycerides 2.1 1.48 4.720.31 Diglycerides 14.9 14.62 20.02 0.81 Triglycerides 46.9 48.9 40.3Free sterols 3.1 1.5 5.2 0.2 FFA 7 5 5 3 Glycerol N.D. N.D. 2.6 N.D.Brassicasterol* 0.54 0.46 0.82 N.D. Campasterol* 5.43 4.58 4.93 0.009Stigmasterol* 2.84 3.86 3.25 0.00142 Betasitosterol* 8.9 8.41 8.1 0.166*levels of the different sterols, which can be either esterified ornon-esterified.

All materials were protected from light and odorous products at atemperature not exceeding 25° C.

Tri- and Di-Glyceride Esters of Omega-3 LC-PUFAs (Fish Oil)

The following Tables 2A, 2B and 2C give the fatty acid and glyceridescomposition of the commercial fish oil used in the LC-PUFA controlgroups in the Examples 1-3 presented below. TABLE 2A Fatty acidcomposition summary (%) DHA 51.1 EPA 23.3 Omega-3 81.6 Oleic acid 3.8

TABLE 2B Fatty Acid Composition (mg/g) C14 2.8 C15 0.3 C16 6.1 C16:1 3.8C17 0.7 C18 1.7 C18:1 36.4 C18:2 5.8 C18:3n3 6.3 C18:4 13.3 C20 0.4C20:1 25.1 C20:2 4.5 C20:4n6 10.4 C20:4n3 15.5 C20:5n3 220.7 C22 0.3C22:1 6.5 C22:5n3 47.4 C22:6n3 484.1 C24 C24:1 2.8 Others 53.3

TABLE 2C Glyceride composition Monoglycerides, % w/w 2.52 Digylcerides,% w/w 28.36 Ratio 1,3/1,2 diglycerides 2.9Phytosterol, Tri, Di- and Monoglyceride Esters of Omega-3 LC-PUFAS

The following Tables 3A to 3F give the composition of the omega3-phytosterol esters mixture (composition of the invention) used inExample 4 presented below. This preparation is referred to herein asPreparation A. TABLE 3A Total Fatty acid composition summary (%) DHA50.3 EPA 23.9 Omega 3 81.6 Oleic acid 3.9

TABLE 3B Total fatty Acid Composition (mg/g) C14 2.0 C15 0.2 C16 4.5C16:1 2.9 C17 0.5 C18 2.0 C18:1 29.7 C18:2 4.6 C18:3n3 4.7 C18:4 10.6C20 0.5 C20:1 18.7 C20:2 3.3 C20:4n6 8.9 C20:4n3 12.1 C20:5n3 180.9 C220.2 C22:1 6.5 C22:5n3 39.0 C22:6n3 381.8 C24 C24:1 2.3 Others 42.4

TABLE 3C Total Sterols, % w/w Brassicasterol 0.821 Campesterol 4.93Stigmasterol 3.25 Beta-sitosterol 8.1 Others 1.11 Total 18.211 Freephytosterol, % w/w 5.2 Phytosterol esters % w/w 22.12Values calculated according to average MW of sterols and FA

TABLE 3D Glycerides composition Monoglycerides, % w/w 4.73 Digylcerides,% w/w 20.02 Ratio 1,3/1,2 diglycerides 3.5

TABLE 3E Fatty Acid composition of PE (%) DHA 49.0 EPA 14.8 Omega 3 72.3Oleic acid 7.5 Total unsaturated 91.0 Total saturated 2.4

TABLE 3F Fatty Acid Composition of Phytosterol esters, mg/g phytosterolesters: C14 1.7 C15 0.2 C16 4.1 C16:1 2.6 C17 0.6 C18 2.0 C18:1 28.6C18:2 4.6 C18:3n3 5.1 C18:4 15.0 C20 0.4 C20:1 11.8 C20:2 2.3 C20:4n62.6 C20:4n3 9.7 C20:5n3 56.1 C22 C22:1 2.6 C22:5n3 17.5 C22:6n3 186.5C24 C24:1 0.9 others 25.4Chemical Preparation of Omega-3 Esters Mixture of Glycerides and Sterols

900 g of fish oil (EPAX 20:50, Pronova) and 200 g of soybeanphytosterols (ChoLevel, Fenchem) were mixed together and dried byheating to 110° C. under vacuum. 3.7 g of sodium methoxide were addedafter pressure was carefully brought to atmospheric by nitrogen additionand temperature was reduced to 100° C. After addition of sodiummethoxide, vacuum conditions were restored. Reaction was followed bymeasuring the content of free phytosterols. When free phytosterol leveldropped below 2% w/w, the reaction was stopped by the addition of 4%citric acid solution (50% concentration). Water was removed under thereaction condition and the resulting mixture is filtered. Filtered oilmixture is further bleached to remove soap residues and the bleachedproduct is molecular distilled (170° C., 0.01 mbar), cooled and theproduct (residue) is supplemented with antioxidants.

The composition of the chemically produced preparation is summarized inTable 4. TABLE 4 Monoglycerides, % w/w 1.3 Diglycerides, % w/w 17.5Ratio 1,3/1,2 diglycerides 3.5 Sterols, mg/g: campesterol 46.8stigmasterol 38.9 β-sitosterol 84.4 others 9.1 Total sterols % w/w 17.9Free phytosterols 1.7 Phytosterol esters 28.4 Sodium, ppm 3.1Preparation of Omega-3 Esters Mixture of Glycerides and Sterols(Concentrated)

500 g of fish oil ethyl esters (EE 20:50, ONC) and 500 g of soybeanphytosterols (ChoLevel, Fenchem, Nanjing, China) were mixed together anddried by heating to 110° C. under vacuum. 5 g of sodium methoxide wereadded after pressure was carefully brought to atmospheric by nitrogenaddition and the temperature was reduced to 100° C. After catalystaddition, vacuum conditions were restored. Reaction was followed bymeasuring the content of free phytosterols. When the free phytosterollevel dropped below 4% w/w the reaction was stopped by the addition of4% water. Water is further removed by centrifugation and the resultingsterol esters are treated with silica (Trysil) and filtered. Thefiltered esters are further bleached to remove soap residues and thebleached product is steam deodorized (200° C., 1 hr) cooled andsupplemented with antioxidants. The sodium is measured in final productand is below 5 ppm. 810 gr of the obtained deodorized phytosterol estersare added to 190 g of fish oil (TG 48:25, ONC), to give fish oilenriched with phytosterol esters. The composition of the final mixtureis summarized in Table 5. TABLE 5 Diglycerides, % w/w 6.7Monoglycerides, % w/w 0.7 Sterols, mg/g: Brassicasterol 1.44 Campesterol111.76 Stigmasterol 90.66 β-sitosterol 185.31 Others 11.01 TotalPhytosterols, % w/w 40.2 Free phytosterols, % w/w 3.5 Phytosterolesters, % w/w 64 Fatty Acid Compostion, mg/g: C14 1.19 C16 4.00 C16:11.75 C18 4.26 C18:1 10.23 C18:2 1.49 C18:3n3 0.73 C18:4n3 1.55 C20 2.97C20:1 17.25 C20:2 2.00 C20:4n6 7.01 C20:4n3 9.28 C20:5n3 143.85 C22 1.39C22:1 15.01 C22:5n3 33.78 C22:6n3 204.41 C24:1 7.00 Others 38.10MethodsAnimal StudiesFree Radical Scavenging Capacity

The free radical-scavenging capacity of olive oil, oliveoil+phytosterols, and PSE+DAG in olive oil was analyzed by the DPPHassay. DPPH (1,1-diphenyl-2-picryl-hydrazyl) is a radical-generatingsubstance that is widely used to monitor the free radical scavengingabilities (the ability of a compound to donate an electron) of variousanti-oxidants [Belinky, P. A. et al. (1998) Free Radic. Biol. Med. 24:1419-29]. The DPPH radical has a deep violet color due to its impairedelectron, and radical scavenging can be followed spectrophotometricallyby the loss of absorbance at 517 nm, as the pale yellow non-radical formis produced. 15 μl from stock solution of each sample were mixed with 1mL of 0.1 mmol DPPH/L in ethanol and the change in optical density at517 nm was continuously monitored.

Isolation of Mouse Peritoneal Macrophages

Mouse peritoneal macrophages (MPM) were harvested from the peritonealfluid of the E^(o) mice (15-25 g) 4 days after intraperitoneal injectionof thioglycolate (24 g/L) in saline (3 mL). Cells (10-20×10⁶/mouse) werewashed 3 times with PBS and re-suspended to 10⁶/mL in DMEM containing 5%fetal calf serum (heat-inactivated at 56° C. for 30 min), 100 Upenicillin/mL, 100 μg streptomycin/mL, and 2 mM glutamine. The cellsuspension was plated into culture dishes and incubated in a humidifiedincubator (5% CO₂, 95% air) for 2 hours. The dishes were washed oncewith DMEM to remove non-adherent cells, and monolayers were incubatedunder similar conditions for 18 hours. Mouse peritoneal macrophages wereisolated from 6 mice from each group, pooled and analyzed in duplicateor triplicate for each assay.

Macrophage Superoxide Release

The production of superoxide anion (O₂—) by mouse peritoneal macrophageswas measured as the superoxide dismutase-inhibitable reduction ofcytochrome C [Yanagitani Y. et al. (1999) Hypertension 33:335-9]. Cells(1×10⁶/well) were incubated in 1 mL of HBSS containing acetyl cytochromeC (80 μmol/L). Superoxide production by the cells was stimulated by theaddition of phorbol myristate acetate (PMA; 0.5 μg/mL) for 1 hour. Tosome control samples, superoxide dismutase (SOD, 30 mg/L) was added. Theamount of superoxide release was determined in the medium and wasexpressed as nmoles of superoxides/mg cell protein, using an extinctioncoefficient of E₅₅₀=21 mmol/L⁻¹cm⁻¹.

Macrophage Peroxide Content

Cellular peroxide levels were determined by flow cytometry usingDichlorofluorescin-diacetate (DCFH-DA) [Goupy, P. et al. (2003) Fr.Journal of Agricultural and Food Chemistry 51(3):615-622]. DCFH-DA is anon-polar dye that diffuses into the cells. In the cells it ishydrolyzed into the nonfluorescent derivate 2′,7′-DCFH, which is polarand trapped within the cells. Under oxidative stress, DCFH is oxidizedto DCF (2′,7′-dichlorofluorescein), which is a fluorescent compound.Peritoneal macrophages (2×10⁶) were incubated with 2.5×10⁻⁵ mol/LDCFH-DA for 30 minutes at 37° C. Reaction was stopped by washes with PBSat 4° C. Cellular fluorescence was determined with a flow cytometryapparatus (FACS-SCAN, Becton Dickinson, San Jose, Calif., USA).Measurements were done at 510 to 540 nm after excitation of cells at 488nm with an argon ion laser.

Serum Lipids Profile

Serum samples were analyzed for their lipid profile including totalcholesterol and triglycerides, by using commercially available kits(Roche Diagnostics, Penzberg, Germany).

Serum Lipids Peroxidation

Serum was diluted 1:4 in PBS. Serum susceptibility to oxidation wasdetermined by incubating serum sample with 100 mM of the free radicalgenerating compound, 2′-2′-azobis 2′-amidinopropane hydrochloride(AAPH), which is an aqueous soluble azo compound that thermallydecomposes to produce peroxyl radicals at a constant rate. The formationof thiobarbituric reactive substances (TBARS) and of lipid peroxides wasmeasured and compared to serum that was incubated under similarconditions, but without AAPH.

PON1 Activity Measurements

PON 1 activity in serum was determined by measuring arylesteraseactivity, using phenylacetate as the substrate. Initial rates ofhydrolysis were determined spectrophotometrically at 270 nm. The assaymixture included 1.0 mM phenylacetate and 0.9 mM CaCl₂ in 20 mM TrisHCl, pH 8.0. Non-enzymatic hydrolysis of phenylacetate was subtractedfrom the total rate of hydrolysis. The E₂₇₀ for the reaction was 1,310M⁻¹ cm⁻¹. One unit of arylesterase activity is equal to 1 μmol ofphenylacetate hydrolyzed/min/ml. Purified enzyme has nearly 2000 unitsof arylesterase activity per mg protein.

Macrophage Oxidative Status

Cellular oxidative stress was examined in DCF-loaded macrophages byflow-cytometry using the conversion of non-fluorescent DCFH-DA to itsfluorescent counterpart DCF as an index.

Macrophage-Mediated Oxidation of LDL

MPM were incubated with LDL (100 μg of protein/mL) for 18 hours, underoxidative stress (in the presence of 2 μmol/L of CuSO₄), after which theextent of LDL oxidation was determined by the TBARS assay.

Macrophage Uptake of Oxidized LDL

MPM were incubated with ¹²⁵I-labeled oxidized LDL (10 μg of protein/ml),and lipoprotein cell-association and degradation by these cells wasdetermined. Lipoprotein cellular degradation was measured in thecollected medium as the trichloroacetic acid (TCA)-soluble, non-lipidradioactivity, which was not due to free iodide. Lipoprotein degradationin a cell-free system was measured under identical conditions, and wassubtracted from the total degradation. The remaining cells were washedthree times with cold PBS and dissolved in 0.1 N NaOH for protein andcell-associated lipoproteins determination.

Statistical Analyses

Student t-test was used for statistical analysis of the results.

Human Studies

These studies were designed as a randomized, single-blind, cross-overclinical intervention trial. These experiments involved testing theeffects of dietary matrices containing specific fatty acid and/or plantsterol mixtures included in normal diets, compared to non-supplementeddiets, on circulating lipid levels, in moderately overweight subjectswith elevated serum lipid levels for whom dietary modification is theprimary and at times only therapeutic recommendation.

Patients

Twenty-four (24) volunteers (11 male, 13 female, age 30-65 yr) withLDL-cholesterol concentrations>130 mg/dL were recruited for this study.Body mass indices (BMI) of subjects ranged from 23-32 kg/m², except forthree subjects in which BMI was between 21.4 and 23 kg/m². Twenty-onevolunteers completed the study (11 male, 10 female).

Diets

All volunteers underwent a feeding trial according to a semi-randomizedLatin square cross-over design containing three dietary phases, eachfour week in duration. The control diet was provided first, then theother phases randomized. Each feeding period was separated by a 4-weekwashout interval, during which volunteers consumed their typical dietswithout restriction. The composition of the diets was similar withrespect to the food and nutrient content. The basic diet contained 30%of energy as fat (see control diet), 80 mg cholesterol/1000 kcal, 12 gfiber/1000 kcal, 15% energy as protein and 55% as carbohydrates. Thevariable component was the treatment oil. Diets were designated as:

-   Control group: baseline plant sterol level of about 200 mg/day,    where the dietary fat was comprised at 70% energy as oleic    acid-enriched vegetable oil.-   PSE+DAG group: 1.7 g/day soy sterols esterified to olive oil fatty    acids (predominantly oleic acid), contained in a diglyceride (DAG)    and triglyceride containing olive oil (total amount=9 g/d).-   LC-PUFA group: 7.6 g/day of fish oil providing the same amount of    EPA and DHA as the plant sterol-fish oil ester diet.-   Omega-3-esters mixture group: 1.7 g/day soy sterols esterified to    fish oil fatty acids (including EPA and DHA), contained in a    diglyceride and triglyceride containing fish oil (total amount=9    g/d)

All meals were equicaloric and breakfast was consumed each day undersupervision. The treatment oil was given within the breakfasts consumedevery day at the clinic under supervision. Volunteers were instructed toeat and drink only materials given to them by the Clinical NutritionResearch Unit (McGill University, Montreal, Canada), except for water.Study volunteers were encouraged to maintain their usual level ofphysical activity. Energy requirements were estimated using the Mifflinequation and multiplied by an activity factor of 1.7%. Energy intakeswere adjusted over the initial 2-week period to maintain constant bodyweight and kept constant for the remaining 4 weeks and the othertreatment phases.

Blood Lipid Analyses

On days 1, 2, 28 and 29, blood samples were obtained in the fastingstate. On day 28, subjects reported at the clinic 4 hours after theyconsumed their normal experimental breakfast so that a blood sample wasobtained in the postprandial state. Plasma was immediately separated andstored at −80° C. until analysis. The general lipid profile (Total, HDLand LDL cholesterol as well as triglycerides) was measured in samplestaken on days 1, 2, 28 and 29 of each phase. Plasma total cholesteroland triglyceride in plasma, and HDL sub-fractions were determined byautomated methods in duplicate on an Abbott Spectrum CCX Analyzer(Abbott, Dallas, Tex.) utilizing enzymatic reagents (Abbott A-GENT). LDLcholesterol was calculated by the Friedewald equation. If triglycerideswere higher than 400 mg/dL, then LDL cholesterol levels were directlymeasured in plasma samples (N-geneous LDL-C assay, Equal Diagnostics),using a method in which LDL precipitated by the dextran/magnesiumsulfate method in order to separate them from HDL. Apolipoproteins A andB100 were measured by nephelometry in samples from days 1, 2, 28 and 29.Lipoprotein (a) was measured by nephelometry in samples from days 1 and28.

Plasma thiobarbituric acid reactive substances (TBARS) content, as amarker of its susceptibility to oxidative stress was determined using acommercial kit (TBARS assay kit, OXI-tek).

Plasma leptin levels were determined at beginning and end of each phaseusing a radioimmunoassay. TNF-alpha and IL-6 levels determined using aradioimmunoassay with monoclonal antibodies. Plasmenogen ActivatorInhibitor-1 (PAI-1) and fibrinogen analysis is carried out by two stageenzymatic assay and with use of a Behring BN-100 nephelometer. Levels ofC-reactive protein (CRP) were assessed using a sensitive immunoassaywith monoclonal antibodies coated to polystyrene beads. Prostatespecific antigen (PSA) levels were determined using a radioimmunoassay.Plant sterol levels of plasma and red cells were determined by gasliquid chromatography. Fatty acids profiles of plasma triglycerides wereassessed using gas liquid chromatography (GLC). Fat soluble vitaminlevels were determined by high performance chromatography (HPLC).

Example 1

Antioxidative Effect of Olive Oil, Olive Oil+Phytosterols and PSE+DAG inOlive Oil Against Macrophage Lipid Peroxidation in E^(o) Mice

As mentioned above, oxidative stress is involved in the pathogenesis ofatherosclerosis. Atherosclerosis is associated with lipids peroxidationof plasma LDL and in arterial cells, including macrophages [Aviram M.(2000) Free. Radic. Res. 33:S85-97]. Under oxidative stress, macrophageperoxide levels become increased, and macrophages generate reactiveoxygen species, leading to their increased atherogenicity [Aviram M.(2000) id ibid].

The apolipoprotein E deficient (E⁰) mice are widely used as an animalmodel for atherosclerosis as they develop severe hypercholesterolemiaand atherosclerotic lesions on a chow diet. Moreover, in E⁰ mice,accelerated atherosclerosis is associated with increased lipidperoxidation of plasma lipoproteins and arterial cells [Keidar S. (1998)Life Sci. 63:1-11].

Angiotensin II (Ang-II), a vasoconstrictor produced by therenin-angiotensin system, has been implicated in atherosclerosis. Ang-IIactivates macrophage NAD(P)H-oxidases, leading to increased macrophagelipid peroxidation [Rajagopalan S. et al. (1996) J. Clin. Invest.97:1916-1923; Johnston R. B. Jr. (1984) Methods Enzymol. 105:365-9].

In the present Example, the anti-oxidative effect of three preparationsof olive oil, designated olive oil+phytosterols, PSE+DAG in olive oiland olive oil, against macrophage oxidative stress was analyzed.

The following oil samples were tested (all diluted in water 1/2 v/v,stock solution):

1. Olive oil+phytosterols

2. PSE+DAG in olive oil

3. Olive oil

The antioxidative effect against macrophage oxidative stress of thePSE+DAG in olive oil in comparison to olive oil+phytosterols and oliveoil alone was analyzed by two parameters: (i) the ability to decreasemacrophage peroxide content; and (ii) macrophage ability to releasesuperoxide ions.

Mouse peritoneal macrophages were incubated with 50 □μl of stocksolution/ml of either olive oil+phytosterols, PSE+DAG in olive oil andolive oil alone for 15 min, followed by a further incubation for 1 hourwith Angiotensin II (10⁻⁷ M) to induce oxidative stress. Control cellswere incubated with Angiotensin II alone. Macrophages were then analyzedfor their peroxides content using the DCFH assay and for their abilityto release superoxide ions (FIG. 2A, B).

1) Effect of Olive Oil, Olive Oil+Phytosterols, and PSE+DAG in Olive Oilon Macrophage Peroxide Content

Pre-incubation of the macrophages with both olive oil+phytosterols andPSE+DAG in olive oil but not with olive oil alone reduced the macrophageperoxide content compared to control macrophages incubated withAngiotensin II alone. The macrophage lipid peroxides content, using theDCFH assay, is assessed by two parameters; first, the mean fluorescenceintensity emitted by DCF and second, the percentage of cells that arepositive for fluorescence emission. Preincubation of macrophages with 50μl/ml of olive oil+phytosterols or PSE+DAG in olive oil led to areduction of 83% and 64% in macrophage mean fluorescence intensitycompared to control cells, whereas olive oil at the same concentrationhad no effect on the macrophage mean fluorescence intensity compared tocontrol cells (FIG. 2A). Similarly, preincubation of macrophages with 50μl/ml of olive oil+Phytosterols or PSE+DAG in olive oil led to areduction of 74% and 55% in percentage of positive cells forfluorescence compared to control cells, whereas olive oil at the sameconcentration had no effect on the percentage of positive cells forfluorescence compared to control cells (FIG. 2B).

2) Effect of Olive Oil, Olive Oil+Phytosterols and PSE+DAG in Olive Oilon Macrophages Superoxides Ions Release

Mouse peritoneal macrophages isolated from E° mice were pre-incubatedwith 50 μl/ml of either PSE+DAG in olive oil, olive oil+phytosterols orolive oil alone for 15 minutes followed by a further incubation for 1hour with Angiotensin II (10⁻⁷ M) to induce oxidative stress. Controlcells were incubated with Angiotensin II alone.

All three olive oils preparations analyzed in the present studyinhibited to some extent, macrophage superoxide release induced byAngiotensin II. However, PSE+DAG in olive oil and olive oil+phytosterolswere significantly more potent than olive oil alone. Pre-incubation ofmacrophages with 50 μl/ml of PSE+DAG in olive oil, oliveoil+phytosterols or olive oil alone led to a reduction of 29%, 23% andonly 9% respectively in macrophage superoxides anions release, comparedto control cells incubated with Angiotensin II alone (FIG. 3).

Olive oil preparations enriched with phytosterols, in particular PSE+DAGin olive oil, exhibited significant anti-oxidative properties againstmacrophage lipid peroxidation. In contrast, whereas olive oil alone didnot exhibit any effect. Most importantly, the PSE+DAG in olive oilcomposition was more potent than the olive oil+phytosterols preparationin its ability to reduce macrophage peroxide content and macrophagesuperoxide release.

These results suggest that olive oil and the additional components(phytosterols and diglycerides) can bind and internalize into themacrophages. In addition, olive oil enrichment with phytosterols enablesthe preparation of the invention to inhibit cellular oxidative systems(such as the NADPH oxidase and/or lypoxygenases) or to activate cellularanti-oxidant systems (such as the glutathione or superoxide dismutasesystems). Furthermore, the addition of DAG to the olive oil+phytosterolpreparation (resulting in the PSE+DAG in olive oil, in accordance withthe invention) led to an additional antioxidative effect towardsmacrophage lipid peroxidation. The inventors thus speculate that DAG,which participates in numerous intracellular signal transductionpathways, could further affect the above cellularoxidative/antioxidative systems which are involved in AngiotensinII-mediated cellular oxidative stress, expressed as macrophage lipidperoxidation and superoxide release.

Example 2

The effect of PSE+DAG in canola oil and PSE+DAG in fish oil on theatherogenicity of lipoproteins and macrophages, and on atherosclerosisdevelopment in the atherosclerotic apolipoprotein E deficient (E^(o))mice model was investigated. Apolipoprotein E deficient (apoE^(o)) miceat 8 weeks of age were assigned randomly to the following groups (5 miceeach) as described below. The mice received regular chow diet, and inaddition, they were fed (via gavage) the following, once every threedays:

Group I:

1. Placebo group: did not receive any addition of oil.

2. Canola oil group (control): were fed with 60 μl of canola oil.

3. PSE+DAG in canola oil group: were fed with 60 μl of PSE+DAG in canolaoil.

Group II:

1. Placebo group: did not receive any addition of oil.

2. PSE+DAG in fish oil: were fed with 60 μl of PSE+DAG in fish oil.

Each mouse consumed approximately 5 mL of water/day, and 5 g ofchow/day.

Oil Preparation for Feeding

The amounts of PSE+DAG in canola oil and PSE+DAG in fish oil fed to themice were based on the following:

The recommended phytosterols dosage for humans is 1.5 gr ofphytosterols/day. Based on 18.1% phytosterols in each sample, the dosagefor PSE+DAG in canola oil and PSE+DAG in fish oil for humans istherefore 1.5/0.18=8.33 gr/day/person. For mice, the body weight shouldbe taken into consideration (60,000 gr human body weight/20 gr mousebody weight=3000), thus the daily dosage for mouse is 8.33 gr/3000=2.78mg/day/mouse, which is equal to 2.78/0.93=2.99 mL/day/mouse. Since theexperiment was done for a limited period, the dosage used was 5-foldhigher. Thus, each mouse was administered 15 mL of oil/day (60 mL/4days/mouse).

At the end of the experimental period, blood samples were collected fromall mice for serum separation and analyses. Within each experimentalgroup, the blood sample of each mouse was analyzed individually. Thefollowing parameters were analyzed in the serum:

1. Determination of lipids, including total cholesterol andtriglycerides levels.

2. Determination of serum oxidative status.

3. Determination of paraoxonase, measured as arylesterase activity.

MPM were harvested prior to removal of the heart and aorta. The micewere anesthetized with ethyl ether in a local nasal container.

The experimental protocol (No. IL-066-10-2001) was approved by theAnimal Care and Use Committee of the Technion Israel Institute ofTechnology (Haifa, Israel).

FIG. 3 shows that the consumption of PSE+DAG in canola oil resulted in aremarkable and significant reduction in the levels of triglycerides inthe serum (36%), in comparison with placebo (p<0.001).

Similarly, FIG. 4 shows that particularly PSE+DAG in canola oildemonstrated a tendency to reduce total cholesterol levels in the serum(p<0.1).

FIG. 5 shows that PSE+DAG in canola oil treatment resulted in a drasticand highly significant (p<0.001) reduction of the serum susceptibilityto AAPH-induced oxidation by 63% (in comparison to placebo). PSE+DAG infish oil demonstrated a similar tendency, reducing lipid peroxides by16% in comparison with placebo. The different efficacy between these twomatrices could be attributed, at least in part, to the well documentedtendency of these polyunsaturated fatty acids towards generation oflipid peroxidation products.

FIG. 6 shows an interesting result. Whereas canola oil consumptioninduced a significant reduction in the levels of serum PON1 activity(p<0.1), which may be detrimental for atherosclerosis [Mackness, B. etal. [2003] Circulation 107:2775-9], consumption of PSE+DAG in canola oilor in fish oil restored PON1 activity, to levels comparable of that ofuntreated (Placebo group) mice. Thus, consumption of PSE+DAG in canolaoil and PSE+DAG in fish oil is beneficial for maintaining effectivelevels of PON1 activity.

FIG. 7 demonstrates that consumption of PSE+DAG in canola oil causedreduction of ox-LDL association (16%) and degradation (14%) (p<0.05),resulting in increased MPM abilities to sustain ox-LDL, which can becorrelated to a decreased oxidation status, and to a larger extent,PSE+DAG in fish oil displayed a similar effect, also causing reductionof ox-LDL association (34%) and degradation (30%) (p<0.001). Incontrast, canola oil consumption resulted in a slight increased (pvalue<0.05) of both ox-LDL association and degradation (4% and 11%,respectively, in comparison to placebo).

FIG. 8 shows that consumption of either PSE+DAG in canola oil or PSE+DAGin fish oil significantly reduced the oxidative status of E⁰ micemacrophages (p<0.0001). PSE+DAG in fish oil reduced macrophagesoxidative status by 34% in comparison to placebo, while PSE+DAG incanola oil reduced it by 29% in comparison to placebo. Thus, bothPSE+DAG in fish oil and PSE+DAG in canola oil are effective in reducingthe oxidative status of macrophages. Consistent with these results, inFIG. 9, the inventors show that, similarly, consumption of eitherPSE+DAG in fish oil or PSE+DAG in canola oil also significantly reducedthe PMA-induced release of superoxide anions in macrophages (p<0.05).

Example 3 Human Study Comparing PSE+DAG Diet with Control

Change in Total Cholesterol Concentrations

Supplementation of mildly overweight hypercholesterolemic subjects' dietwith PSE+DAG demonstrated a distinct impact on total plasma cholesterolconcentrations (P-value<0.0001; see FIG. 10). Compared to control MUFAbased OA-enriched oil diet, PSE+DAG consumption induced a significantlyhigher hypocholesterolemia effect (−4% vs. −12%, respectively; P=0.03).In terms of absolute cholesterol concentrations reduction, thevolunteers that consumed these diets reduced 13 mg/dL and 30 mg/dL; frominitial average level of 240 mg/dL and 243 mg/dL to a final averagelevels of 228 mg/dL and 213 mg/dL, respectively. It must also be notedthat this background diet, contained limited amount of cholesterol (notmore than 80 mg/1000 kcal), which could also play part in the totalcholesterol levels reduction effect. With respect to the American HeartAssociation (AHA) recommendations towards blood lipids levels [ExpertPanel on Detection, Evaluation, and Treatment of High Blood Cholesterolin Adults (2001) J.A.M.A. 285:2486-2497], suggesting the desirable totalcholesterol levels in healthy individuals to be below 200 mg/dL, thevolunteers who consumed the vegetable-oil diet reduced 31% of the totalcholesterol levels required to meet the target. However, followingPS-E+DAG feeding the serum total cholesterol levels were markedlyreduced towards these optimal level inasmuch as 71%.

Change in LDL-Cholesterol Concentrations

As shown in FIG. 11, the hypocholesterolemic effect of PS-E+DAG was moreprominent, as expected, in the reduction of LDL cholesterol plasmalevels (from baseline levels of 154±8 mg/dL to endpoint 135±7 mg/dL;P-value=0.0004; while control vegetable oil consumption resulted in asignificant effect on reduction of LDL cholesterol concentrations;however to a somewhat lower extent (from baseline levels of 156±8 mg/dLto endpoint 148±7 mg/dL; P-value=0.051; see FIG. 11). Indeed, in apaired analysis of the percentages of change in the LDL-cholesterolconcentrations after supplementation with the PSE+DAG mixture comparedto those observed after control oil supplementation, a marked tendencywas demonstrated (−11.91% versus −4.13%, respectively; P=0.058). Thesehypercholesterolemic overweight volunteers, fed for four weeks withcontrol MUFA based OA-enriched oil diet or PSE+DAG followed by fourweeks of washout and counter supplementation, reduced their blood LDL-clevels by 31% or 81%, respectively, with respect to AHA definition ofLDL-C levels of 130 mg/dL as near to optimal levels.

Change in apoB Concentrations

Lipoproteins and their associated apolipoproteins, like LDL-c and apo B,reduction was reported following OA-enriched olive oil diet, as comparedto high omega-6 enriched diet in diabetic type 2 patients [Madigan C.,et al., (2000) Diabetes care 23:1472-1477]. Therefore, a significantreduction in these volunteers' apo B levels following base oilconsumption was expected (from baseline values of 1.19±0.07 g/L toendpoint 1.13±0.05 g/L; P-value=0.016; FIG. 12). As previously shown forplant sterol esters spreads [Amundsen A L., et al. (2002) Am. J. Clin.Nutr. 76:338-344], a more pronounced effect was demonstrated followingPS-E+DAG supplementation to these hypercholesterolemic volunteers (frombaseline values of 1.21±0.06 g/L to endpoint 1.09±0.06 g/L;P-value=0.0002; FIG. 12). Moreover, a comparison of the reducing effectof PSE+DAG on endpoint apo B concentrations (−9.85%) to the one obtainedby the control diet (−2.77%), suggested a statistically significantdifference between these dietary treatments effects (P=0.022).Interestingly, Kondo and colleagues [Kondo A., et al. (2002) J.Atheroscler. Thromb. 9:280-287] suggested a strong correlation betweenoxidized-LDL and apo B concentrations, having a turning point at apo Blevels above 1.15 g/L. Both diets, were shown to be beneficial inobtaining this anti-oxidant outcome, albeit PSE+DAG presenting a morepotent effect.

Change in apoB/apoA Ratio

Apo B, apo A-I and the apo B/apo A-I ratio have been reported as betterpredictors of cardiovascular events than LDL-C and they even retaintheir predictive power in patients receiving lipid-modifying therapy[Walldius J. and Jungner I. (2004) J. Intrn. Med. 255:188-205]. As maybe seen from FIG. 13, in addition to the aforementioned reduction in apoB levels, introducing PSE+DAG to the base MUFA-enriched diet resulted inmarked and significantly reduced levels of apoB/apoA ratios (frombaseline values of 0.89±0.05 to endpoint 0.84±0.05; P-value=0.047; whileno comparable effect was obtained following control diet (from baselinevalues of 0.91±0.04 to endpoint 0.89±0.04; P-value=0.19; FIG. 13). Nosignificant differences were observed between the effects of thesetreatments on apoB/apoA ratios in % change (−4.01% and −1.35%,respectively; P=0.22). Similar observations were reported followingplant sterol or stanol esters spreads consumption [Amundsen A L., et al.(2002) id ibid; Hallikainen M A. et al. (2000) J. Nutr. 130:767-776].

Change in Lipoprotein (a) Levels

As shown in FIG. 14, hypercholesterolemic volunteers fed with controldiet presented significantly elevated levels of Lp(a) (from baselinevalues of 0.18±0.03 g/L to endpoint 0.22±0.04 g/L; P-value=0.0003; FIG.14). However, esterification of monounsaturated fatty acids to plantsterols in a DAG containing matrix (PS-E+DAG) maintained Lp(a)concentrations (from baseline values of 0.19±0.03 g/L to endpoint0.19±0.03 g/L; P-value=0.41; FIG. 14).

Change in Triglycerides Levels

Recently, an elevated triglycerides concentration was suggested to be aunivariate predictor of CVD. Specifically, 88 mg/dL increase intriglycerides levels was associated with a 14% increase in CVD risk inmen and a 37% increase in women [Austin M A., et al. (1998) Am. J.Cardiol. 81:7B-12B]. Olive oil based diet was already demonstrated toinduce a marked (10%) triglycerides levels reduction, comparing with anaverage American diet [Kris-Etherton P M., et al., (1999) Am. J. Clin.Nutr. 70:1009-1015]. Therefore, the reduction in the fastingtriglycerides levels of the different diets was anticipated, as the basediet oil was OA-enriched (control diet from 191±32 mg/dL to 165±25mg/dL, P-value=0.038 and PSE+DAG in olive oil 182±27 mg/dL to 155±22mg/dL, P-value=0.031). The extent of the PSE+DAG effect on fastingtriglycerides levels (−9.9%) was at least somewhat higher than what wasobtained for control high oleic diet (−5.3%), which could also beappreciated from the difference in the median values of % of change(−10.4% and −3.7%, respectively). It should be also noted that thetested subjects were normolipidemic but presented elevated levels ofLDL-C. It is well established [Normen L., et al. (2004) Curr Med ChemCardiovasc Hematol Agents 2:1-12] that even potent dietaryhypotriglyceridemic agents like fish oil demonstrate different effect innormolipidemic as opposed to hyperlipidemic subjects.

Change in Oxidative Stress Levels

Aviram and Eias [Aviram M. and Eias K. (1993) Ann. Nutr. Metab.37:75-84] had demonstrated that in vitro incubation of LDL with copperion in the presence of linoleic acid (LA) resulted with a 22% elevationof LDL oxidation, as opposed to marked dose-dependent inhibition oflipoprotein oxidation by pre-incubation with OA. Moreover, a recentstudy [Madigan C., et al., (2000) Diabetes care 23:1472-1477] suggestedthat, in type 2 diabetes, an OA-rich Mediterranean-type diet versus aLA-enriched diet may reduce the risk of atherosclerosis by decreasingthe number of chylomicron remnant particles. In consistence, controldiet, enriched with MUFA, demonstrated a mild tendency toward reducingthe oxidative levels, as recorded using TBARS analysis (from baselinevalues of 1.11±0.48 mmol/L to endpoint 0.91±0.11 mmol/L; P-value=0.10;FIG. 15). Importantly, following PSE+DAG consumption there was a morepronounced and significant reduction in plasma oxidative stress levels(from baseline values of 1.01±0.21 mmol/L to endpoint 0.83±0.15 mmol/L;P-value=0.005; FIG. 15). These observations could be attributed in partto the aforementioned obtained indications for marked reduction of totalcholesterol, LDL-C, apo B, and triglycerides while maintaining Lp(a)levels, which were previously shown to be correlated with elevated serumanti-oxidant capacity levels.

Example 4 Human Study Comparing Preparation a Diet (Omega-3 Esters) withFish Oil

Change in Total Cholesterol Concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol diet (open squares) fish oil (closed squares) or Preparation A(hatched squares) followed by four weeks of washout and countersupplementation. Total cholesterol levels were tested at the beginningand at the termination of each phase as described in methods. Valuesrepresent mean±SEM of the percent of change in the total cholesterolconcentrations in 21 patients. Statistical significance between thedifferent treatments as found by ANOVA is P<0.05.

FIG. 16 presents the effects of the dietary treatments on plasma totalcholesterol concentrations in all patients (n=21) in the differentphases. A substantial decrease (−8.45%; p-value=0.008) in totalcholesterol concentrations was observed following the fish oiltreatment, while control diet consumption induced a significant thoughsmaller hypocholesterolemic effect (−4.40%; P-value=0.03). In terms ofabsolute cholesterol concentrations reduction, the volunteers thatconsumed these diets reduced 21 mg/dL and 13 mg/dL; from initial averagelevel of 241 mg/dL and 240 mg/dL to a final average levels of 220 mg/dLand 228 mg/dL, respectively.

Change in LDL-Cholesterol Concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withfish oil (open squares) or Preparation A (closed squares) followed byfour weeks of washout and counter supplementation. LDL-cholesterollevels were tested at the beginning and at the termination of each phaseas described in methods, and the results are shown in FIG. 17. Valuesrepresent mean±SEM of the percent of change in LDL-cholesterolconcentrations in 21 patients. Statistical significance between the fishoil and Preparation A as found by paired Student's t-test analysis isP<0.05.

As shown in FIG. 17, the hypocholesterolemic effect of Preparation A ofthe invention was more prominent, as expected, in the reduction of LDLcholesterol plasma levels (FIG. 17); while fish oil consumption resultedin an elevation of LDL cholesterol concentrations, compared to thereduction in LDL cholesterol obtained by the control diet. Theesterification of fish oil with plant sterol esters attenuated theincreasing effect of fish oil on LDL cholesterol (from 158 mg/dL to 144mg/dL; P-Value=0.006). The percentages of change in the LDL-cholesterolconcentrations after supplementation with the omega-3 esters mixture ofthe invention (Preparation A) were statistically different from thoseobserved after fish oil supplementation (−8.04% versus −0.93%,respectively; P<0.05). A paired analysis of Preparation A effect onLDL-cholesterol compared to the control diet had demonstrated a markedtendency (8.04% versus 4.13%; P-value=0.15).

Change in apoB Concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol diet (open squares) fish oil (closed squares) or Preparation A(hatched squares) followed by four weeks of washout and countersupplementation. Apolipoprotein B100 (apoB) levels were tested at thebeginning and at the termination of each phase as described in methods.The results are shown in FIG. 18. Values represent mean±SEM of thepercent of change in the apoB concentrations in 21 patients. Statisticalsignificance between the different treatments as found by ANOVA isP<0.05.

As shown in FIG. 18, a comparison of the reducing effect of the omega-3esters mixture of the invention (Preparation A) on endpoint apoBconcentrations (−12.52%) to the ones obtained by un-reacted fish oil(−8.84%) or control diet (−2.27%), suggests a statistical significantbetween these dietary treatments effects (P=0.012).

Change in apoA/apoB Ratio

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol diet (open squares) fish oil (closed squares) or Preparation A(hatched squares) followed by four weeks of washout and countersupplementation. Apolipoprotein B100 and Apolipoprotein A levels weretested at the beginning and at the termination of each phase asdescribed in methods. The results are shown in FIG. 19. Values representmean±SEM of the percent of change in the apoA to apoB ratio in 21patients. Statistical significance between the different treatments asfound by ANOVA is P<0.01.

As may be seen from FIG. 19, in addition to the change in apoB levels,when the omega-3 esters mixture of the invention (Preparation A) werecompared to the fish oil and control diets, significant differences wereobserved between the effects of treatment on apoA/apoB ratios absolutechanges (0.09, −0.01 and 0.02, respectively; P=0.039) and % change(8.45%, 0.35% and 2.20%, respectively; P=0.09) in apoA/apoB ratios.

Change in Total to HDL Cholesterol Ratio

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol diet (open squares) fish oil (closed squares) or Preparation A(hatched squares) followed by four weeks of washout and countersupplementation. Total cholesterol and HDL-cholesterol levels weretested at the beginning and at the termination of each phase asdescribed in methods. The results are shown in FIG. 20. Values representmean±S.E.M of the percent of change in the total cholesterol toHDL-cholesterol ratio in 21 patients. Statistical significance betweenthe different treatments as found by ANOVA is P<0.01.

As shown in FIG. 20, fish oil supplementation resulted in a tendency toincrease the total/HDL cholesterol ratio (+3.5%; P-Value=0.15),apparently related to a decrease in HDL cholesterol levels (−10.94%;P-value=0.00003), while the control diet demonstrated only a negligiblean insignificant increase in this key parameter (0.05; P-Value=0.72).However, when the omega-3 esters mixture of the invention (PreparationA) was consumed with the control diet, a significantly decreased (7.5%;P-Value=0.008) total/HDL cholesterol ratio was demonstrated (compared tofish oil and control diet; P-value=0.005; FIG. 20).

Changes in HDL Cholesterol Subfractions

Hypercholesterolemic mildly overweight volunteers were fed for fourweeks with control diet (open squares) fish oil (closed squares) orPreparation A (hatched squares) followed by four weeks of washout andcounter supplementation. HDL₂ (A) and HDL₃ (B) cholesterol levels inblood were measured at the beginning and at the termination of eachphase as described. Results are shown in FIG. 21. Values representmean±SEM of % of change in HDL₂ or HDL₃-cholesterol sub-fraction in 21patients. Statistical significant as found by ANOVA for A (P-value<0.02)and B (P-Value<0.005).

As shown, feeding hypercholesterolemic subjects for a limited periodwith either control diet or fish oil had no effect on the HDL₂subfraction levels (7.8% and −6.1%, respectively; P-value=0.33 and0.45). However, supplementation with the omega-3 esters mixture of theinvention (Preparation A) resulted with a marked elevation in HDL₂levels (38.7%; P-value=0.007), which was shown to be significantlydifferent than the control diet or fish oil treatment effects(P-value=0.022; FIG. 21A). The HDL₃ levels were reduced similarly byeither the omega-3 esters mixture of the invention (Preparation A) orthe fish oil (−13.1% and −12.7%, respectively; P-Value of 0.002 and0.0005), but were unaffected by the control diet consumption (0.40%;P-Value=0.49), as shown in FIG. 21B.

Change in Fasting Triglycerides Concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol diet (open squares) fish oil (closed squares) or Preparation A(hatched squares) followed by four weeks of washout and countersupplementation. Triglycerides levels were tested at the beginning andat the termination of each phase as described in methods. Results areshown in FIG. 22. Values represent mean±SEM of the level of plasmatriglycerides concentrations in 21 patients. Statistical significancebetween the different treatments as found by ANOVA is P<0.0001.

Un-reacted fish oil decrease plasma fasting triglycerides by 37.1% (from178 mg/dL to 97 mg/dL; P-value=0.007) while the comparable effect of theomega-3 esters mixture of the invention (Preparation A) was 42.9%decrease (from 165 mg/dL to 87 mg/dL; P-Value=0.00002); however therewas only a mild tendency that suggests a difference between the effectsof these supplementations (Student's t-test Paired analysis theP-Value=0.112). Comparing the effects of these supplements on fastingtriglycerides levels to the rather mild effect of the control diet(−5.3%; P-Value=0.04) provides a statistically significant differencebetween the consumption of fish oil to olive oil (P-Value=0.0001; seeFIG. 22).

Endpoint Postprandial Triglycerides Concentrations.

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol diet (open squares) fish oil (closed squares) or Preparation A(hatched squares) followed by four weeks of washout and countersupplementation. Triglycerides levels were analyzed shortly after themeal at the termination of each phase as described in methods. Resultsare shown in FIG. 23. Values represent mean±SEM of the level ofpostprandial triglycerides concentrations in 21 patients. Statisticalsignificance between the different treatments as found by ANOVA isP<0.01.

FIG. 23 shows the effect of the diets on postprandial triglyceridelevels (FIG. 23). The lowest plasma triglyceride concentrations afterconsumption of a meal were detected in the subjects fed with the omega-3esters mixture of the invention (135.6 mg/dL) compared with fish oil(159.1 mg/dL) and control diet (232.6 mg/dL) supplements(P-Value=0.002). Interestingly, the paired analysis between theun-reacted and the omega-3 esters mixture of the invention (PreparationA) demonstrated a notable tendency between these two supplements, thoughthe levels of the EPA and DHA were identical (P-Value=0.09).

Change in CRP Concentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol diet (open squares) fish oil (closed squares) or Preparation A(hatched squares) followed by four weeks of washout and countersupplementation. CRP levels were analyzed at the beginning and thetermination of each phase as described in methods. Results are shown inFIG. 24. Values represent mean±SEM of the level of CRP concentrations in20 patients. Statistical significance between the different treatmentsas found by ANOVA is P<0.15.

The comparison of short-term consumption of control diet with or withoutfish oil or the omega-3 esters mixture of the invention (Preparation A)presented only a mild tendency towards altered effects (P-Value=0.151;see FIG. 24).

Endpoint Relative Risk According to Total/HDL Cholesterol and CRPConcentrations

Hypercholesterolemic overweight volunteers were fed for four weeks withcontrol diet (open squares) fish oil (closed squares) or Preparation A(hatched squares) followed by four weeks of washout and countersupplementation. Total cholesterol, HDL cholesterol and CRP levels wereanalyzed at the termination of each phase as described in methods. Totalto HDL cholesterol ratio was calculated as described in methods, andRifai and Ridker 2000 [Rifai and Ridker (2001) Clin. Chem.; 47:28-30]suggested algorithm was employed relative risk in the different phasescalculation. Results are shown in FIG. 25. Values represent mean±SEM ofthe relative risk predicted values according to Rifai and Ridker 2000 ascalculated for 21 patients. Statistical significance between the controldiet or fish oil and the Preparation A as found by paired Student'st-test is 0.004 and 0.005, respectively.

Further correlations between the total to HDL-cholesterol ratio andCRP-levels remarkably indicate a reduced relative risk of CVD eventprospective in the following ten years for the subjects which consumedthe omega-3 esters mixture of the invention (Preparation A) (FIG. 25).

1. An edible mixture of esters of long chain polyunsaturated fatty acids(LC-PUFA), said mixture comprising esters of LC-PUFA with phytosterolsand/or phytostanols and esters of LC-PUFA with glycerol, optionally alsocomprising free phytosterols and/or phytostanols, wherein the weightratio of said LC-PUFA glycerol esters to their esters with phytosterolor phytostanol is from about 19:1 to about 1.5:1.
 2. A mixture of claim1, wherein said esters of LC-PUFA with glycerol are mono-, di- and/ortriglycerides.
 3. The mixture of claim 1, for use in reducing the riskof developing metabolic disorder and/or insulin resistance syndrome. 4.The mixture of claim 1, for use in reducing circulating atherogenicsmall-dense LDL particles, while maintaining a high proportion ofHDL/LDL ratio.
 5. The mixture of claim 1, for use in shifting HDLprofile in the direction of larger and less dense particles,particularly reducing the HDL₃ subfraction while increasing the level ofHDL₂ subfraction.
 6. The mixture of claim 1, for use in reducing and/orpreventing the progression of the metabolic disorder and/or insulinresistance syndrome and/or atherosclerotic deleterious processes.
 7. Themixture of claim 1, for use in attenuating or counteracting any adverseeffects of use of omega-3 fatty acids in the form of oils.
 8. Themixture of claim 7, wherein said adverse effect may be elevating LDLcholesterol or increasing the total/HDL cholesterol ration, related to adecrease in HDL cholesterol levels.
 9. An edible composition of mattercomprising a mixture of esters of LC-PUFA with glycerol, enriched with amixture of esters of LC-PUFA with phytosterols and/or phytostanols,wherein said mixture of esters LC-PUFA with glycerol is derived from anyone of animal source, particularly fish oil, plant, algae andmicroorganisms, and wherein the weight ratio of said LC-PUFA glycerolesters to their esters with phytosterol or phytostanol is from about1.5:1 to about 1:9.
 10. The composition of matter of claim 9, for use inreducing the risk of developing metabolic disorder and/or insulinresistance syndrome.
 11. The composition of matter of claim 9, for usein reducing circulating atherogenic small-dense LDL particles, whilemaintaining a high proportion of HDL/LDL ratio.
 12. The composition ofmatter of claim 9, for use in shifting HDL profile in the direction oflarger and less dense particles, particularly reducing the HDL₃subfraction while increasing the level of HDL₂ subfraction.
 13. Thecomposition of matter of claim 9, for use in reducing and/or preventingthe progression of the metabolic disorder and/or insulin resistancesyndrome and/or atherosclerotic deleterious processes.
 14. Thecomposition of matter of claim 9, for use in attenuating orcounteracting any adverse effects of use of omega-3 fatty acids in theform of oils.
 15. The composition of matter of claim 14, wherein saidadverse effect may be elevating LDL cholesterol or increasing thetotal/HDL cholesterol ration, related to a decrease in HDL cholesterollevels.
 16. An orally administrable pharmaceutical or nutraceutical unitdosage form comprising an edible mixture of esters LC-PUFA withglycerol, enriched with a mixture of esters of LC-PUFA with phytosterolsand/or phytostanols, wherein said unit dosage form contains about1.4-1.6 g of said phytosterol/phytostanol LC-PUFA esters, said dosageform being capable of providing at least 50% of the recommended dailyintake (RDI) of phytosterols and the recommended daily intake ofLC-PUFA.
 17. The pharmaceutical dosage form of claim 16, in the form ofa capsule, preferably a soft gel capsule.
 18. The pharmaceutical dosageform of claim 16, wherein said RDI of phytosterols is from about 0.4 gto about 0.8 g and said RDI of LC-PUFA, specifically, DHA and EPA, isfrom about 0.3 g to about 0.65 g.
 19. The pharmaceutical dosage form ofclaim 16, for use in reducing the risk of developing metabolic disorderand/or insulin resistance syndrome.
 20. The pharmaceutical dosage formof claim 16, for use in reducing circulating atherogenic small-dense LDLparticles, while maintaining a high proportion of HDL/LDL ratio.
 21. Thepharmaceutical dosage form of claim 16, for use in shifting HDL profilein the direction of larger and less dense particles, particularlyreducing the HDL₃ subfraction while increasing the level of HDL₂subfraction.
 22. The pharmaceutical dosage form of claim 16, for use inreducing and/or preventing the progression of the metabolic disorderand/or insulin resistance syndrome and/or atherosclerotic deleteriousprocesses.
 23. The pharmaceutical dosage form of claim 16, for use inattenuating or counteracting any adverse effects of use of omega-3 fattyacids in the form of oils.
 24. The pharmaceutical dosage form of claim23, wherein said adverse effect may be elevating LDL cholesterol orincreasing the total/HDL cholesterol ration, related to a decrease inHDL cholesterol levels.
 25. The pharmaceutical dosage form of claim 16,for twice daily administration.
 26. A method of treatment of metabolicsyndrome and related conditions, said method comprising administering atherapeutically effective dose of an edible mixture of esters of LC-PUFAor compositions comprising thereof to a subject in need, wherein saidmixture comprises esters of LC-PUFA with phytosterols and/orphytostanols and esters of LC-PUFA with glycerol, wherein the weightratio of said LC-PUFA glycerol esters to their esters with phytosterolor phytostanol is from about 19:1 to about 1:9.
 27. The method of claim26, wherein said effective dose is comprised in a soft gelatine capsule,said method comprising administering said capsule to said patient twicedaily.
 28. A method of reducing circulating atherogenic small-dense LDLparticles, while maintaining a high proportion of HDL/LDL ratio, saidmethod comprising administering a therapeutically effective dose of anedible mixture of esters of LC-PUFA or compositions comprising thereofto a subject in need, wherein said mixture comprises esters of LC-PUFAwith phytosterols and/or phytostanols and esters of LC-PUFA withglycerol, wherein the weight ratio of said LC-PUFA glycerol esters totheir esters with phytosterol or phytostanol is from about 19:1 to about1:9.
 29. The method of claim 28, wherein said effective dose iscomprised in a soft gelatine capsule, said method comprisingadministering said capsule to said patient twice daily.
 30. A method ofshifting the HDL profile in the direction of larger and less denseparticles, particularly reducing the HDL₃ subfraction while increasingthe level of HDL₂ subfraction, said method comprising administering atherapeutically effective dose of an edible mixture of esters of LC-PUFAor compositions comprising thereof to a subject in need, wherein saidmixture comprises esters of LC-PUFA with phytosterols and/orphytostanols and esters of LC-PUFA with glycerol, wherein the weightratio of said LC-PUFA glycerol esters to their esters with phytosterolor phytostanol is from about 19:1 to about 1:9.
 31. The method of claim30, wherein said effective dose is comprised in a soft gelatine capsule,said method comprising administering said capsule to said patient twicedaily.